Functional dissection of the S. cerevisiae
helicase activating factor Cdc45
by
Sze Ham (Bena) Chan
B.S. Biochemistry, BME
Tufts University, 2009
Submitted to the Department of Biology
In partial fulfillment of the requirements for the degree of
Master of Philosophy in Biology
MASSXCHUETTS INST1I1fE
.O TECHNOLGY
at the
FT,
1 2023
Massachusetts Institute of Technology
L i7RARIES
June 2013
@2013 Sze Ham
ena) Chan. All rights reserved.
The author hereby grants to MIT the permission to reproduce and distribute
publicly paper and electronic copies of this thesis in whole or in part.
Signature of Author:_________________
Department of Biology
June 30, 2013
Certified by:
U-,
Stephen P. Bell
Professor of Biology
Thesis Supervisor
Accepted by:
Stephen P. Bell
Professor of Biology
Co-Chair, Committee for Graduate Students
a
Functional dissection of the S. cerevisiae helicase activating factor Cdc45
by
Sze Ham (Bena) Chan
Submitted to the Department of Biology on June 30, 2013 in partial fulfillment of the
requirements for the degree of Master of Philosophy in Biology
ABSTRACT
Eukaryotic DNA replication initiation requires two essential steps: helicase loading
and activation. During the G1 to S transition, loaded Mcm2-7 helicases are activated
by the S-CDK (S phase cyclin-dependent kinase)- and DDK (Dbf4-dependent kinase)dependent recruitment of the helicase activating proteins Cdc4S and GINS. The
resulting Cdc45/Mcm2-7/GINS (CMG) complex unwinds double-stranded DNA at
the replication fork. Although it is clear that CMG complex possesses robust helicase
activity in vitro that is dependent on GINS and Cdc45, it is unclear how the CMG is
assembled in vivo. Moreover, the mechanism of Cdc45 and GINS stimulation of
Mcm2-7 helicase activity is unclear. In budding yeast, recruitment of Cdc45 and
GINS occurs sequentially with Cdc45 associating with Mcm2-7 in a DDK- and Sd3dependent manner. Subsequently, S-CDK stimulates the association of GINS in a
Sd2-, DNA Pol epsilon- and Dpbll-mediated event.
To better understand the mechanism of helicase activation, I have dissected the
function of Cdc45 using a combination of in vivo and in vitro approaches. I generated
a series of deletion and site-specific mutations of Cdc45 based on homology to RecJ,
structural prediction algorithms and sequence conservation. Functional domain
mapping of Cdc45 revealed that both N- and C-terminal segments are essential in
vivo. Analysis of site-specific mutations identified five lethal and three temperaturesensitive site-specific mutations. Three of these mutations are within a putative
DHH phosphoesterase domain related to bacterial RecJ, suggesting a critical role for
the RecJ-homology region for Cdc45 function. Two lethal site-specific mutations
were found within a predicted intrinsically disordered region (IDR). Oddly, a
complete deletion of the IDR is dispensable for Cdc45 function in vivo.
Mutants that failed to fully complement a CDC45 deletion in vivo were purified and
tested for their ability to complement Cdc45 function biochemically. Purified Cdc45
mutants were found to be associated with Hsp70 chaperones, and subsequently
failed to fully restore in vitro replication in a Cdc45-dependent biochemical assay.
Co-immunoprecipitation studies revealed an interaction of Cdc45 with Sd3 and
S1d7 and mapped a Cdc45 region that interferes with the Sld7 interaction. Together,
these preliminary data provide a starting point for future mechanistic studies to
understand role of Cdc45 in helicase activation.
Thesis Supervisor: Stephen P. Bell
Title: Professor of Biology
3
Acknowledgments
First and foremost, I would like to thank my advisor Professor Bell for his patient
guidance and step-by-step supervision. Steve is inspirational to my development as
a scientist and has provided me with immense help in overcoming my missteps. This
thesis would not have been possible without his timely and valuable advice. I also
thank members in Bell lab for their kind assistance and technical expertise, and for
creating a homely, intellectually stimulating work environment.
I want to acknowledgment my thesis committee Professor Orr-Weaver and
Professor Cheeseman for their feedback on my project. Besides, I wish to express
my gratitude to Professor Walker and Professor Solomon, for their generous
support and encouragement. I also want to give my heartfelt thanks to my
classmates Miao and Theresa, for their company in my time of need and for many
wonderful memories.
Finally, I am forever indebted to my parents and my husband Ruoshi, to whom this
thesis is dedicated. Their endless love, care and understanding are the source of my
strength throughout my life.
4
Table of Contents
A b stra ct...............................................................................................................................3
Acknow ledgments..............................................................................................................4
Table of Contents......................................................................................................
5
Chapter I: Introduction............................................7
Overview of eukaryotic replication initiation......................................................8
Helicase activation during G1/S transition.................................................
10
Eukaryotic replicative helicase: CM G helicase com plex..............................
17
The core M cm 2-7 helicase................................................................
18
Helicase activator GINS.......................................................................
23
Helicase activator Cdc45.....................................................................
28
Structure of CM G com plex...................................................................
35
Thesis Sum m ary..............................................................................................
37
References.......................................................................................................
38
Chapter II: Role of the S. cerevisiae helicase activator protein Cdc45...........48
Sum m ary.........................................................................................................
49
Introduction.....................................................................................................
50
Results..................................................................................................................54
Mutagenesis of Cdc45................................................................................
54
In vivo complementation analysis of Cdc45 mutants.............................
59
Purification of Cdc45 mutant proteins...................................................
65
Co-im m unoprecipitation of Cdc45, S d3 and S1d7.................................
70
In vitro Cdc45-dependent replication assay................................................
74
5
Discussion.......................................................................................................
81
Cdc45 as an intrinsically disordered protein.........................................
81
Hsp70 interaction w ith m utant Cdc45 proteins....................................
82
Role of the N-term inus of ScCdc45...........................................................
86
Evolutionary link betw een Cdc45 and RecJ............................................
88
Role of the C-term inus of ScCdc45.....................................................................
89
Cdc45 interaction w ith Sld3 and Sld7......................................................
89
Potential function of the Cdc45 IDR ...............................................................
92
Future characterization of Cdc45 m utants............................................
93
M aterials and M ethods...................................................................................
94
Supplem ental Material...................................................................................
96
References.......................................................................................................
98
6
Chapter I
Introduction
7
Overview of eukaryotic DNA replication initiation
Eukaryotic DNA replication initiation is precisely coordinated in space and time so
that the genome is duplicated exactly once per cell cycle. It is important that each
copy of intact chromosome is faithfully duplicated before chromosome segregation
and cell division. Over- or under-replication caused by defects in the assembly or
regulation of the replication machinery causes genomic instability, cancer and cell
death (reviewed in Blow & Gillespie, 2008).
To ensure eukaryotic chromosomes are replicated completely, replication initiation
events are temporally separated into two non-overlapping phases: helicase loading
and activation (reviewed in Bell & Dutta, 2002; Sclafani & Holzen, 2007; Remus &
Diffley, 2009) (Figure 1). In the model eukaryote S. cerevisiae,cell cycle-regulated
kinases, S-phase cyclin-dependent kinase (S-CDK) and Dbf4-dependent kinase
(DDK), are critical to this control.
During G1 phase when S-CDK and DDK activity is low, the eukaryotic replicative
helicase called Mcm2-7 complex is loaded at potential sites of replication initiation
known as origins of replication. Helicase loading is initiated by binding of origin
recognition complex (ORC) at origin DNA (Bell & Stillman, 1992). During G1, ORC
recruits Cdc6 followed by a complex between Cdtl and the hetero-hexameric
Mcm2-7 helicase. Together these proteins load the Mcm2-7 complex as a
catalytically inactive, head-to-head double-hexamer that encircles double-stranded
DNA (dsDNA)(Evrin et al, 2009; Remus et al, 2009; Gambus et al, 2011).
8
Figure 1
Control of eukaryotic DNA replication initiation
Helicse Le~irgH
elc ase Activa tjon
(GI)
S-CDK
(S G2 M)
Hgh CDK Ac#vity
Origin firing
Origin recognition
CDK
No helicase
activation
No new helicase
loading
Figure 1. S. cerevisiaereplication initiation events are temporally separated
into two non-overlapping phases by CDK regulation: helicase loading and
activation. CDK has both positive and negative roles for eukaryotic replication
initiation. In absence of high CDK activity, helicase loading is allowed during late M
to G1 phase (in pink). At this stage, Mcm2-7 helicases can be loaded as inactive,
head-to-head double hexamers that encircle double-stranded DNA. Upon entry into
S phase, increased CDK and DDK activities lead to recruitment of helicase activating
proteins to a subset of Mcm2-7 (in orange). The resulting active Mcm2-7 complex
translocates on single-stranded DNA that unwinds DNA to provide template for
replisome assembly. In budding yeast, de novo helicase loading outside G1 phase is
inhibited by CDK phosphorylation via several mechanisms. CDK regulation ensures
origin firing once and only once per cell cycle.
9
During the G1 to S transition, increased S-CDK and DDK activity activates a subset of
the loaded Mcm2-7 helicases. Origin DNA unwinding by active helicases is required
for replisome assembly and DNA synthesis, leading to the establishment of a pair of
bi-directional replication forks. Upon entry into S phase, re-replication is prevented
by multiple, distinct pathways that inhibit de novo helicase loading until the next G1
phase (reviewed in Labib K, 2010; Remus & Diffley, 2009).
Helicase activation during G1/S transition
Despite many similarities in replisome architecture between prokaryotic and
eukaryotic DNA replication, the mechanism of helicase activation is distinct. In E.
coli, the replicative helicase DnaB is loaded on single-stranded DNA (ssDNA)
(Biswas & Biswas-Fiss, 2006). DnaB is subsequently activated through a
DnaG/primase-dependent event that triggers the release of the helicase
loader/inhibitor DnaC (Makowska-Grzyska et al, 2010). The activated DnaB
hexamer travels along the lagging strand template in a 5' to 3' direction (reviewed in
McGlynn P. 2011).
The process of eukaryotic helicase activation is more complicated and less well
understood. This event must transform the loaded Mcm2-7 from an inactive,
double-hexameric, dsDNA-encircling state to an active, single-hexameric, ssDNAencircling-state. This transformation must disrupt the Mcm2-7 double-hexamer
interface and extrude one DNA strand from each Mcm2-7 central channel (reviewed
in Boos et al. 2012) (Figure 2). The molecular events triggering and
10
Figure 2
Eukaryotic helicase activation:
Transition from inactive to active helicase
MCM
V6
Double hexamer
encircling dsDNA
Inactive Helicase
S-CDK & DDK:
GINS & Cdc45
Separation of double hexamer
Strand extrusion
recruitment
IP
Mcm2-7
S
W-dc45
QINS
Active Helicase: CMG
Single hexamer on
ssDNA 3 to 5'
Figure 2. Eukaryotic helicase activation involves the transition from a Mcm2-7
double-hexamer encircling dsDNA to a CMG complex encircling ssDNA. The
loaded Mcm2-7 double hexamer encircles dsDNA and is inactive. The result of
helicase activation is formation of Cdc45-Mcm2-7-GINS (CMG) complex, which
functions as a ssDNA translocase that actively unwinds DNA duplex at a replication
fork. To achieve this transition, separation of the Mcm2-7 double hexamer and
extrusion of ssDNA would be required.
11
during the transformation remain elusive. By the end of eukaryotic helicase
activation, two helicase activating factors, Cdc45 and GINS, are tightly associated
with each Mcm2-7 to form the Cdc45/Mcm2-7/GINS (CMG) complex. The CMG
complex is the active helicase that travels on ssDNA along the leading-strand
template in a 3' to 5' direction (Ilves et al. 2010; Fu et al. 2011). This direction is
opposite to the polarity of the prokaryotic DnaB helicase. The ssDNA generated is
coated and stabilized by ssDNA-binding replication protein A (RPA) (Tanaka &
Nasmyth, 1998; Walter &Newport, 2000). Unwinding of origin DNA is required for
complete replisome assembly, most notably the recruitment of Pol a/primase to the
origin DNA [Heller ref, and bi-directional DNA synthesis by replicative polymerases
(Kamimura et al. 2001; Nakaya et al. 2010).
In budding yeast S. cerevisiae,activation of the Mcm2-7 helicase requires both S-CDK
and DDK activity (reviewed in Labib K. 2010; Li &Araki, 2013; Heller et al. 2011).
These kinases modify Mcm2-7 and other replication initiation proteins to drive the
origin association of multiple replication initiation factors. The most important of
these are the two helicase activators (Cdc45 and GINS) that triggers origin firing
(Figure 3).
Multiple lines of evidence suggest DDK phosphorylation induces a conformational
change in Mcm2-7 that facilitates helicase activation. Both in vivo and in vitro studies
demonstrate that DDK docks and phosphorylates mainly the Mcm2, 4 and 6
subunits of loaded Mcm2-7 (Randell et al, 2010; Sheu &Stillman, 2006; Francis et al;
12
Figure 3
AMPre-RClhelase
kloed
M00000(y.
rQCdO
DDK 2
S
00K-dependent
Ib
complex
*f
OPb11 g~I
CE)K
r
IV
Complete
Figure 3. Model for helicase activation and replisome assembly in S. cerevisiae.
Mcm2-7 helicase is first loaded at a replication origin in an inactive state. The
subsequent helicase activation requires both DDK and S-CDK activity. Recruitment
of a Sld7-Sld3-Cdc45 complex to the loaded Mcm2-7 is DDK-dependent. CDK acts
after DDK, facilitates recruitment of Sld2, Dpbll, GINS, and Pol E to Mcm2-7. After
chromatin association of both Cdc45 and GINS, replication factor McmlO associates.
Active DNA unwinding is required for recruitment of the lagging strand DNA
polymerases to complete replisome assembly. (Figure adapted and modified from
Heller etal. 2011)
13
2009). In budding yeast, it has been suggested that the only role of DDK
phosphorylation is to alleviate an inhibitory activity of N-terminal serine/threoninerich domain (NSD) of Mcm4 (Sheu &Stillman, 2010). Removing the Mcm4 NSD
bypasses essential DDK function. Moreover, a mutation in Mcm5 (bob-1 allele) can
mimic DDK phosphorylation and allow constitutive loading of Cdc45 in vivo
(Sclafani et al. 2002; Hoang et al. 2007), even though Mcm5 itself is not a DDK target
(Randell et al, 2010). These DDK bypass studies suggest that DDK phosphorylation
plays a regulatory role that changes Mcm2-7 conformation. Since DDK stimulates
origin recruitment of S1d3 and Cdc45 (Heller et al. 2011; Kamimura et al. 2001;
Aparicio et al. 1999), conformational changes of the Mcm2-7 complex are likely
required for the association of these factors. Recently, a newly identified factor Sld7
has been found to directly and constitutively interact with Sld3 (Tanaka T. et al.
2011). This Sld3-Sld7 complex associates with Cdc45 in late G1 and together is
recruited to early-firing origins in a DDK-dependent manner (Tanaka S. et al. 2011).
Upon entry into S phase, S-CDK, acts after DDK (Heller et al. 2011; Walter JC. 2000),
to drive helicase activation by phosphorylating Sld2 and Sld3 (Tanaka et al. 2007).
These phosphorylation events result in the origin-DNA recruitment of another
helicase activator-GINS (Heller et al. 2011; Takayama et al. 2003). It has been
shown in a bypass study that phosphorylation of Sd2 and S1d3 is the only essential
function of CDK during yeast DNA replication initiation (Tanaka S. et al. 2007;
Zegerman & Diffley, 2007). CDK phosphorylation of Sd2 and S1d3 generates
phosphopeptides that bind to the N-terminal pair (BRCT1-2) and C-terminal pair
14
(BRCT 3-4) of Dpb11 BRCT repeats, respectively (Tak et al. 2006; Zegerman &
Diffley, 2007; Tanaka S, 2007).
There is evidence that phospho-Sld2/Dpbll/GINS and DNA Pol E,associate prior to
their origin recruitment to the origin in a S-CDK-dependent manner (Muramatsu et
al. 2010). In line with this finding, a biochemical study in budding yeast indicates
that the N-terminal domain of Pol E subunit Dpb2 directly interacts with GINS and is
required for the CMG assembly during replication initiation in vivo (Sengupta et al.
2013). A recent study using yeast two-hybrid and co-IP shows that GINS interacts
with inter-BRCT (between BRCT 2 and BRCT 3) region of Dpb11 (Tanaka S et al.
2013). Taken together, it seems that CDK phosphorylation delivers the second
helicase activator GINS and the leading strand polymerase DNA Pol E to originassociated Cdc45 and loaded Mcm2-7, by promoting a molecular bridge between
Cdc45-phospho-Sld3, Dpbll, and phospho-Sld2. In times of replication stress and
DNA damage, S phase checkpoints are activated that target Sld3 and Dbf4 to block
late origin firing (Lopez-Mosqueda etal., 2010; Zegerman & Diffley, 2010). Sld3
phosphorylation mediated by the checkpoint kinase Rad53 inhibits its interaction
with both Cdc45 and Dpb11 (Zegerman & Diffley, 2010).
Although multiple replication initiation factors must be recruited to the origin to
drive helicase activation, most are not required for ongoing helicase activity. Sld2,
Sld3 and Dpbll are each released from the chromatin prior to replication
(Kanemaki & Labib, 2006; Masumoto et al, 2000). In vitro interaction studies
15
propose that Sld3 release could be the result of GINS recruitment competing for the
sameor overlapping binding sites on Mcm2-7 (Bruck &Kaplan, 2011).
Mcm1O has also been implicated in helicase activation. Mcm1O is essential for DNA
replication, but unlike Cdc45 and GINS, it is not a stable component of replication
fork (reviewed in Thu &Bielinskey, 2013). There are conflicting reports as to
whether Mcm1O is involved in assembly, activation or remodeling of CMG complex.
The discrepancies might be due to different experimental methods. Xenopus and
fission yeast studies suggest that Mcm1O is recruited to chromatin before DDK in G1
phase and Mcm1O is required for origin association of Cdc45 (Wohlschlegel et al.
2002; Gregan et al. 2003). In a human cells study using bimolecular fluorescence
complementation (BiFC) for monitoring CMG association, it was reported that
Mcm1O and accessory factors such as Ctf4 are required for CMG assembly (Im et al.
2009). However, several recent studies in yeast and human cells support a model in
which Mcm1O is recruited to chromatin after CMG assembly (Heller et al 2011;
Kanke et al. 2012; van Deursen et al. 2012; Watase et al. 2012). These studies
suggest that Mcm1O is required for initial DNA unwinding in vivo. The authors find
that when Mcm1O is depleted or inactivated, RPA (the ssDNA binding protein)
association with the origin DNA is reduced (an indication of less ssDNA and
therefore less DNA unwinding) but recruitment of Cdc45 and GINS is not affected
(Kanke et al. 2012; van Deursen et al. 2012; Watase et al.2012). Caution needs to be
exercised when interpreting these results, however, as RPA association is not a
16
direct measurement of DNA unwinding activity and chemical crosslinking in ChIP
assays might enrich loosely bound Cdc45 (reviewed in Thu & Bielinsky, 2013).
Besides a potential role in DNA unwinding, Mcm1O is also thought to be required for
recruitment of lagging strand polymerases (Pol a and Pol 6) either directly or
indirectly (Heller et al. 2011). Consistently, multiple studies indicate that Mcm1O
binds to the catalytic subunit of Pol a and is required for Pol a chromatin association
(Warren et al. 2009; Ricke & Bielinsky, 2004; Yang et al. 2005; Zhu et al. 2007).
Intriguingly, ubiquitination of Mcm1O leads to its release from Pol a, and allows it to
directly interact with PCNA. The consequence of the ubiquitinated Mcm1O-PCNA
interaction is unclear, but this interaction is essential in budding yeast (Das-Bradoo
et al. 2006). Mcm1O might play a dual role in DNA unwinding and DNA polymerase
recruitment, making it an attractive candidate for being a factor that coordinates
helicase and polymerase activity at the replication fork.
Eukaryotic replicative helicase: the CMG complex
The CMG complex is the eukaryotic replicative enzyme that catalyzes unwinding of
double-strand DNA at replication forks. It is an eleven-member complex composed
of the hexameric MCM and the helicase activators Cdc45 and GINS (a tetramer).
The CMG complex was first isolated from extracts of early Drosophilaembryos
through purification of Cdc45 using traditional and immunoaffinity methods (Moyer
et al. 2006). Unlike Mcm2-7 alone, which little or no helicase activity, purified CMG
complex is sufficient for robust helicase activity in vitro (Bochman & Schwacha,
2008; Ilves et al. 2010). This suggests that Cdc45 and GINS activate Mcm2-7 by
17
stimulating its ATPase activity. Recent in vitro characterization of the purified
human CMG using baculovirus Sf9 system shows that hCMG prefers forked DNA
structures with 3'-tail. Increasing the length of 3'-ss tail significantly increases both
helicase and DNA-binding activities (Kang et al, 2012). In an in vitro rolling circle
assay, the hCMG unwinds DNA duplex region up to 500bp and longer regions in
presence of ssDNA binding proteins (Kang et al, 2012). Similar to DrosophilaCMG
complex, hCMG requires ATP and magnesium to bind to ssDNA and hydrolyzes ATP
in absence of DNA (Kang et al, 2012, Ilves et al. 2010). Consistent with the CMG
being the replicative helicase, each component of the complex has been shown to
travel with the replication fork in vivo (Aparicio et al. 1997; Kanemaki & Labib,
2006) and is required for ongoing fork movement both in vivo and in vitro (Pacek &
Walter, 2004; Kanemaki & Labib, 2006).
The core Mcm2-7 helicase
The catalytic core of eukaryotic replicative helicase is the heterohexameric Mcm2-7
(minichromosome maintenance) complex that is composed of six related, but
functional distinct AAA+ (ATPase associated with a variety of cellular activities)
ATPase subunits (Mcm2, 3, 4, 5, 6, and 7, Iyer et al. 2004; Koonin 1993). The sixpolypeptide subunits associate to form a torroid with a central channel large enough
to accommodate either dsDNA or ssDNA (Pape et al. 2003; Fletcher et al. 2003;
Brewster et al. 2008).
Like other AAA+ proteins, Mcm2-7 ATPase sites are formed at the interface between
18
Figure 4
Hexamer 1
C-terminal
AMA+
Hexamer 2
N-
N-
ter*W ternpW
C0ernwal
Central
channel
2/15
1if
3/7
5/3
A
714
w W
230A
150A tiAs
6/2
4/
Cu.vpo~
0~ in coo sw
C
Iel
Figure 4. MCM structural architecture. A. 3D EM reconstruction of MCM shows
that it forms a double hexamer connected via the N-terminal rings. (Figure adapted
from Boos et al. 2012) B. Organization of Mcm2-7 subunits. ATPase active sites form
at the interface of the subunits. For each pair, one contributes a P-loop formed by
both Walker A and B motifs in cis for ATP binding and hydrolysis; while another
contributes SRF motif of the arginine finger in trans for ATP hydrolysis. (Figure
adapted from Forsburg, 2004) C Schematics of beta hairpins extruding from the
central and side channels, based on the archaeal Mcm crystal structure. The side
channels are wide enough for ssDNA passage. (Figure adapted from Brewster et al.
2010)
19
adjacent subunits. One subunit contributes the cis-actingWalker A and B motifs
(forming a P-loop subdomain) required for ATP binding and hydrolysis, while the
other contributes a trans-acting"arginine finger" (within the conserved SRF motif)
that stimulates hydrolysis of the ATP bound to the adjacent subunit (reviewed in
Bochman &Schwacha, 2009) (Figure 4B). Mutational analysis shows that all six
ATPase sites are essential for viability (Schwacha & Bell, 2001).
Although lacking a high-resolution Mcm2-7 structure, crystal structures of
homohexameric archaeal MCM shed light into eukaryotic Mcm2-7 helicase
structural architecture. EM and X-ray crystallography studies reveal that MCM has a
N-terminal oligonucleotide-binding (OB) fold domain that is thought to facilitate
oligomerization (Bae et al. 2009), and a C-terminal catalytic domain that includes
the conserved AAA+ module. Archaeal MCM can oligomerize into a double hexamer
in solution, with the N-terminal domains associate to form head-to-head doublehexamers with a central channel for DNA to pass through (Fletcher et al. 2003;
G6mez-Llorente et al. 2005; Costa et al. 2006) (Figure 4A).
Archaeal MCM crystal structures also reveal four positively charged
P-hairpins
around the central and side channels that affect DNA binding activity (McGeoch et
al., 2005; Jenkinson &Chong, 2006) (Figure 4C): the NT-hairpin (or NT-hp) is found
within the N-terminal domain while the the exterior hairpin (EXT-hp), the helix 2
insert hairpin (H2I-hp) and the pre-sensor 1 hairpin (PS1-hp) are found within the
C-terminal domain (Figure 5D). The PS1-hp and H21-hp are proposed to undergo
20
Figure 5
A
B
tl
4
1B~AA
H21-hpgo
goo
PS1-hp
D
N
NT-hp
PSI-hp
EXT- )
hp
H21-hp
C 4
Figure 5. Model of near-full length archaeal ssoMCM structure showing four
types of P-hairpins and conformational change with in presence of nucleotide
binding. (Figures of panel A, B &D adapted from Brewster et al. 2010) A. Top and
side views of ssoMCM structure in absence of ATP. B. Proposed model that simulates
conformational change of ssoMCM after closure of nucleotide binding pocket. C.
(Figure adapted from Vijayraghavan & Schwacha, 2012). Arrow heads indicate H21
and PS1 hairpins that undergo rearrangement as discussed in Brewster et al. 2008
and Jenkinson &Chong, 2006. D. ssoMCM monomer (PDB: 3F9V) showing 4 types of
P-hairpins from both N- and C domains.
21
conformational rearrangement following ATP binding (Brewster et al. 2010) (Figure
5A-C). Importantly, mutations within the PS1-hp and H21-hp eliminate helicase
activity (McGeoch et al. 2005; Jenkinson & Chong, 2006).
Eukaryotic Mcm2-7 resemble the archaeal MCM hexamer. Mcm2-7 also forms a
head-to-head double hexamer as detected by 3D-EM and gel filtration analysis. The
double-hexamer encircles dsDNA within its central channel, as visualized by direct
mounting and tungsten rotary shadowing (Evrin et al. 2009; Remus et al. 2009)
However, formation of the Mcm2-7 double hexamer does not occur in solution.
Instead, this form of the enzyme only occurs after loading onto origin DNA (Evrin et
al. 2009; Gambus et al. 2011; Remus et al. 2009). Mcm2-7 loading can be
reconstituted in vitro, and experiments with both linear and circular DNA reveal that
loaded Mcm2-7 is salt-resistant and can slide passively along the DNA in an ATPindependent fashion (Evrin et ei. 2009; Remus et al. 2009).
A mechanistic understanding of Mcm2-7 DNA unwinding remains to be determined.
Recent single-molecule study using strand-specific biotin-streptavidin DNA
roadblocks in Xenopus cell-free extracts shows that CMG is a ssDNA translocase (Fu
et al. 2011). This observation supports a "steric exclusion" model in which the
Mcm2-7 helicase sterically displaces an unengaged strand from its central channel
during replication elongation. However, these findings cannot exclude possible
alternative DNA unwinding modes of Mcm2-7 as a double hexamer. For example,
the single-molecule studies did not analyze the initial melting of origin DNA. How
22
ATP binding and hydrolysis of Mcm2-7 helicase is coupled to DNA unwinding is still
unknown. By analogy to the papillomavirus El protein, it is likely that ATP binding
and hydrolysis by Mcm2-7 is coupled to conformational changes of the previously
described hairpins. Interaction of these hairpins with DNA then drive unidirectional
movement on ssDNA (Enemark & Joshua-Tor, 2006).
Structural studies suggest that Mcm2-7 can alternate between a closed- and an
open-ring conformation. This change would modulate the access of DNA to its
central channel (Bochman & Schwacha, 2008; Davey et al. 2003). It is unclear how
the Mcm2-7 interacts with DNA but it is likely that this interaction involves the
positively charged P-hairpins that protrude from each MCM subunit into the central
channel (Brewster et al. 2008). Single-particle electron microscopy and 3D
reconstructions of purified DrosophilaMcm2-7 reveals that the Mcm2-7 complex is
in equilibrium between a planar, notched conformation and a spiral, lock-washer
conformation (Figure 6A & B). In both Mcm2-7 conformations, a gap is present at
the interface between Mcm2 and Mcm5 (Costa et al. 2011). This structural finding
agrees with previous biochemical studies indicating Mcm2/5 serves as a "gate"
controlling entry of DNA into the central channel of the Mcm2-7 complex (Bochman
& Schwacha, 2007; Davey et al. 2003).
Helicase activator GINS
GINS is an essential, tetrameric complex that is composed of four related but distinct
subunits S1d5, Psfl, Psf2 and Psf3 (named after Japanese 'go-ichi-ni-san') (Takayama
23
Figure 6
a
AAA+
2
Notched
Mcm2-7
Side
) Mcm2
4Wcm4
* Mcm6
b
W
*
Mcn7
*
Mcrn3
TD
0
cm
AAA+
3
Lock-washr
M~cm2-7
Figure 6. Drosophila Mcm2-7 exists in two conformations (notched-ring or
lock-washer) based on single-particle electron microscopy and 3D
reconstruction. The Mcm2-7 complex exists in both a planar, notched
conformation and a spiral, lock-washer conformation in solution. The two
conformations are in equilibrium with about 28% notched form (A) and 72% lockwasher form (B). An obvious gap can be found between Mcm2 and Mcms interface,
especially in the lock-washer form of Mcm2-7. (Figure adapted from Costa et al.
2010)
24
et al. 2003) with a stoichiometry of 1:1:1:1 (Kubota et al. 2003). GINS was
discovered by two independent genetic screens in budding yeast (Kamimura et al.
1998; Kanemaki et al. 2003). SIdS was isolated in a screen for mutations that were
synthetically lethal with Dpb11 (sid stands for synthetic lethality with dpb11-1)
(Kamimura et al. 1998). The remaining subunits were identified in a screen for
essential genes that inhibited replication when mutated (Kanemaki et al. 2003) or as
proteins that interacted with Sld5 while the rest of the GINS complex was identified
as its protein interactors (psf stands for Partner of SId Five) (Takayama et al. 2003).
Consistent with its role as a key component of the CMG helicase, GINS is a part of
replication progression complex (RPC) (Gambus et al. 2006; Pacek et al. 2006) and
is essential for establishment and maintenance of replication fork (Kanemaki et al.
2003; Kanemaki & Labib, 2006). The GINS complex is recruited to chromatin in a Sphase-specific and (Takayama et al. 2003) CDK-dependent manner (Heller et al.
2011). In formaldehyde fixed cell extract, GINS can be detected to be in a complex
with Pol E, Sld2, Dpb11 (Muramatsu et al. 2010).
EM studies of GINS heterotetramer reveals a C-shaped complex with a central cavity
that could encircle ssDNA (Boskovic et al. 2007; Kubota et al. 2003) (Figure 7C).
Other independent crystallographic studies of the human GINS complex describe
GINS as an overall compact, elongated, two-layered structure with a smaller space
for DNA binding (Choi et al. 2007; Kamada et al. 2007; Chang et al. 2007). Sld5 and
Psf1 are found on the upper layer while Psf2 and Psf3 on the bottom layer, with a
25
Figure 7
10 A
AB
'""" "
*
fm
C
IkNib
D
---.-- -Human
Human GINS
S~d5
GINS
CE
Truncated ,
'~
~
P.52P.53
Free Form
N
A-domain
B-domain
A-domtain
B-dman
A-domain
Figure 7. GINS structure. A: Crystal structure of human GINS. (PDB code 2E9X;
Kamada et al. 2007) Psfl in green, Sld5 in yellow, Psf2 in light blue, Pst3 in magenta.
B: Overall GINS structure by molecular surface representation, a narrow central
cleft is shown. C: EM structure of human GINS, showing a C-shaped structure, unlike
crystal structure. (EMDB entry: 1355; Boskovic et al. 2007) D: ribbon drawing of the
solved structures of the four GINS subunits. E: Cartoon drawing the organization
the tetramer. Psfl B-domain is no required for GINS tetramerization. (Figure 7A &
7C adapted from Onesti& McNeill, 2013; Figure 7B, D & E adapted from Kamada,
2012)
26
narrow cavity at the center that seems incapable of accommodating ssDNA (Figure
7A & B). The inconsistency between GINS EM and crystal structures might due to
the limitation of low resolution EM analysis, or a possibility that GINS might adopt
an open or closed conformation reflected by different sample preparation. None of
the GINS crystal structures is complete, as the Psfl C-terminus is truncated because
it is disordered and also sensitive to protease (Choi et al. 2007; Kamada et al. 2007;
Chang et al. 2007). Each GINS subunit is composed of a-helix-rich (A)-domain and Pstrand-rich (B) domain (Figure 7D). The truncated C-terminus of Psf1 is predicted to
the B domain. Although the flexible Psfl B domain is not required for GINS complex
formation (Figure 7E), it is essential for DNA replication in Xenopus egg extracts
(Kamada et al. 2007). Interestingly, a recent study shows that Psf1 B domain of GINS
directly binds to the N-terminus of a Pol E subunit Dpb2. Such an interaction could
coordinating CMG helicase function with leading-strand polymerase action at the
replication fork (Sengupta et al. 2013).
It remains an open question whether GINS can bind DNA. One study reports that
human GINS preferentially binds ssDNA over dsDNA using electrophoretic mobilityshift assay (EMSA)(Boskovic et al, 2007). However, another study of Pyrococcus
furiosus GINS found no DNA binding activity (Yoshimochi et al. 2008). A
reconstituted, mutant DrosophilaCMG lacking the Psf3 C-terminal truncation
reveals a reduced helicase activity and affinity for forked DNA substrates, suggesting
that GINS might directly or indirectly enhance DNA binding ability of CMG complex
(Ilves et al, 2010). There is also biochemical evidence that human GINS interacts
27
with and stimulates Pola-primase for increased DNA synthesis of singly-primed
M13 (De Falco et al. 2007).
Helicase activator Cdc45
Cdc45 was initially isolated in a cold-sensitive cell cycle mutant screen in yeast
(Moir et al. 1982) and was later shown to be required for both the initiation and
elongation stages of DNA replication both in vivo and in vitro (Hopwood & Dalton,
1996; Aparicio et al. 1997; Zou & Stillman, 1998; Tercero et al. 2000; Pacek & Walter,
2004). The importance of Cdc45 is highlighted by the fact that it is essential for
replication and is highly conserved in both sequence and function from yeast to
human (reviewed in Onesti & Macneill, 2013).
Cdc45 contains a canonical bipartite nuclear localization signal (cNLS) that has been
mapped in budding yeast (Hopwood and Dalton, 1996). ScCdc45 is transported into
nucleus through cNLS transporter importin a (Pulliam et al, 2009) and mainly
localized to the nucleus (Hopwood and Dalton, 1996). Although the mRNA level of
Cdc45 peaks during G1/S transition, its protein level stays at a low but constant
level throughout the cell cycle (Owens et al. 1997). Cdc45 was reported to be an in
vitro DDK target (Weinreich & Stillman, 1999), however there is no evidence that
Cdc45 phosphorylation is functionally important (Albuquerque et al. 2008;
Dephoure et al. 2008). Human Cdc45 includes an anaphase promoting
complex/cyclosome (APC/C)-specific destruction boxes. Accordingly, hCdc45 levels
increase in the presence of proteasome inhibitors (Pollok & Grosse, 2007),
28
suggesting hCdc45 degradation might be mediated by APC/C-ubiquitinylation
followed by degradation by the 26S proteasome.
In budding yeast, chromatin binding of Cdc45 can be observed in late G1 at early-
firing origins (Aparicio et al. 1999). Although DDK is traditionally thought to be
inactive in G1, this initial association is dependent on the catalytic subunit of DDK,
Cdc7 [Heller and other refs]. It is likely that the Gi-association of Cdc45 at earlyfiring origins facilitates their preferred selection for initiation as cells enter S phase.
Consistent with this idea, Cdc45 is present at low levels in cells, making it a limiting
factor for replication initiation (Wong et al. 2011; Tanaka S et al. 2011). Origin
recruitment of Cdc45 is dependent on action of DDK, but not S-CDK (Heller et al.
2011; Jares & Blow, 2000; Walter, 2000). After entry into S phase, origin association
of Cdc45 increases significantly (Aparicio et al, 1999; Heller et al. 2011; Broderick et
al. 2012), consistent with its stable association with Mcm2-7 and GINS in a DDKand CDK-dependent manner.
In agreement with Cdc45 being part of the replisome, this protein interacts with
many replication proteins (Bauerschmidt & Pollok, 2007; Zou & Stillman, 2000).
Genetic and biochemical evidence shows that yeast Cdc45 physically interacts with
Sld3, GINS and Mcm2-7 during helicase activation (Ilves et al. 2010; Kamimura et al.
2001; Tanaka et al, 2011; Bruck & Kaplan, 2011). The sites on Cdc45 that mediate
these interactions have not been mapped. EM studies of Mcm2-7 and the CMG
complex (Costa et al. 2011) suggest Cdc45 interacts with S1d5 and Psf2 subunits of
29
GINS, as well as Mcm2 and Mcm5. The individual physical interactions appear to be
weak (Moyer et al. 2006; reviewed in Vijayraghavan &Schwacha, 2012) which
might account for the failure to reconstitute in vitro helicase activity simply by
incubation of Cdc45, Mcm2-7 and GINS together. The assembly of a stable CMG
complex requires additional replication factors such as Sld2, Sld3, Dbp11 and posttranslational modifications in vivo (Heller et al. 2011).
An enzymatic activity for Cdc45 has not been described, but recent fluorescence
anisotropy and EMSA studies in human and yeast showed that Cdc4S binds ssDNA
in a sequence-independent manner (Bruck &Kaplan, 2013; Krastanova et al 2012).
Further studies identified site-specific mutations at the C-terminus of Cdc45 that
disrupt Cdc45 binding to ssDNA. Importantly, these mutants did not alter its Cdc45
interaction with Mcm2-7, GINS or Sld3. The same C-terminal mutant shows
sensitivity to HU and MMS in vivo, suggesting a possible role of Cdc45 in response to
replication stress (Bruck & Kaplan, 2013).
Although homologs of both GINS and MCM proteins have been described in archaea,
no counterpart has been found for Cdc45. Intriguingly, recent bioinformatics
analysis reveals that Cdc45 has a sequence similarity to the bacterial RecJ, which
does have an archaeal counterpart (Sanchez-Pulido et al.2011; Krastanova et al.
2012; Makarova et al. 2012) (Figure 8). RecJ is a metal-dependent, ssDNA
exonuclease that functions in DNA resection during recombination, base excision
repair and mismatch repair (reviewed in Onesti &MacNeill, 2013).
30
Figure 8
090
1"
C414S
kOeJ
CdC4
8.
rkanmi
?thJ charteristic
Gc5-n
the
ReJ
gn
Re-m.t.fs
--------------------
--
I
Redbxan
-nd-------sg
----.yrved-m-t-fs
II01 -
~
4
is
strng
t
naa~~~ca
vta,
-
Z91
(
sh.wn
hC4C4%SI*
RkofeJ
d
tehCond----------hfgn
193
13---------1
1
=
.-
enugh-t-be
t
k
---
-----gs
evetanou------eanss.
0UIM
dtecte304
e
h
~
~ ~
gybased
W
-
=
PA
9
mainly
t
-
V
239
392
Ofo
t - -a-e
-avanrrn...M
IAVLLWPB3-
AUAXLD~bl
- ----
----------
4S&A-----
ICd4S
uA 391
6
Figure 8. Sequence alignment between archaeal Recj/Cdc45, bacterial Rec
and hCdc45. Only the Recj core (domain I and 11) were used in the alignment, with
the characteristic RecJ motifs shown by Red box and conserved motifs in bold. For
the first half of alignment (up to motif IV), the similarity is strong enough between
RecJ and Cdc4S is strong enough to be detected based on sequence alone. The
second half of alignment that lacks high sequence homology is based mainly on foldrecognition/threading algorithms. The unqiue insertion for eukaryotic Cdc45 that
contains charged patches is shown in magenta, while the insertion (with putative
helical structure) that is present in archaeal RecJ/Cdc45 and hCdc45 but absent in
bacterial RecJ is shown in yellow. (Figure adapted from Krastanova et al. 2012)
31
RecJ exonucleases belong to the DHH nuclease superfamily that share four sequence
motifs (I-IV) (Figure 8 &9A). Motif III includes a catalytic triad with the invariant
sequence of DHH. This motif is found to coordinate metal binding and hydrolysis of
phosphodiester bonds (Aravind & Koonin, 1998; Yamagata et al. 2002). Notably, this
consensus motif is mutated in eukaryotic Cdc45 (for example, ScCdc45 RecJ motif III
has DAH instead of DHH), strongly suggesting that Cdc45 is not an exonuclease.
Consistently, despite retaining weak ssDNA binding ability, purified recombinant
human Cdc45 has neither metal binding nor nuclease activity (Krastanova et al.
2012). Whether the RecJ motifs of Cdc45 play a functional role in DNA replication
remains unknown.
Analysis of the crystal structure of full-length T. thermophilus Recj provides
additional insights into possible Cdc45 functions (Yamagata et al. 2002). The
catalytic core (domain I and II) of bacterial Recj can be used to model Cdc45
structure (Figure 9B-D). Using small-angle X-ray scattering, a low-resolution
envelope for a free human Cdc45 was inferred and suggests that Cdc45 has a
compact core flanked by two lateral extensions. The compact core can be
superimposed onto the catalytic core of bacterial RecJ. The two lateral extensions
are predicted to correspond to a pair of unique insertions present in eukaryotic
Cdc45 that are not found in bacterial Recj (Krastanova et al. 2012) (Figure 8 &
Figure 9A, 9D). A higher resolution 3D structure of free Cdc45, or Cdc45 as a part
CMG complex in presence or absence of DNA will be informative for future RecJCdc45 modeling.
32
Figure 9
A
s.ctefial Rec
B
1 1
domain
I
I
domaMn U
C
D
Figure 9. Bioinformatics and structural information on Cdc45 and Recj. A.
Schematics comparing sequences of eukaryotic Cdc45, bacterial and archaeal RecJ.
Domain I and domain II are the catalytic core of bacterial RecJ. Arrows indicate the
positions of the two eukaryotic insertions that are not found in bacterial RecJ. One
insertion is common to eukaryotic Cdc45 and archaeal RecJ/Cdc45. B. Crystal
structure of RecJ catalytic core (PDB: 2ZXP). C. The conserved residues in four DHH
motifs that coordinate two Mn2+ ions shown in magneta. D. A model for human
Cdc45 implied from structural data by small-angle X-ray scattering and
superimposed with crystal structure of RecJ catalytic core. The position of the two
unique insertions were pointed by the arrows. (Figure adapted from Onesti &
MacNeill, 2013)
33
In mammalian cells, Cdc45 is marker for cell proliferation (Pollok et al. 2007) and
overexpression of Cdc45 blocks cell growth (Wong et al. 2011). Cdc4S knockdown
(by RNAi) in human tumor cells induces apoptosis, suggesting that Cdc45 could be a
potential drug target for cancer therapy (Feng et al. 2003). In a study using UVmimetic agent BPDE, it was shown that chromatin association of Cdc45 reduces in a
dose- and time-dependent manner following BPDE treatment accompanied by Chk1
activation and inhibition of DNA synthesis. After low dose BPDE treatment, Cdc45
chromatin association and DNA synthesis is recovered, suggesting Cdc45 might be a
target for Chkl-mediated S-phase checkpoint pathway (Liu et al. 2006). Similar
Cc45 chromatin dissociation kinetics was observed after induction of UVC damage.
However in the same study using in vivo Fluorescence Correlation Spectroscopy
(FCS) and gel filtration analysis, replisomes containing Cdc45 after UVC treatment
had a similar size and distribution compared to controls. These findings indicate
that Cdc45 is still in a large multi-protein complex after DNA damage (Broderick et
al. 2012).
Interestingly, Cdc45 may play a role during chromatin decondensation. It has been
reported that RNAi knockdown of DrosophilaCdc45 results in chromosomecondensation (Christensen &Tye, 2003). In line with this finding, targeting Cdc45 to
a chromosomal locus promotes large-scale decondensation of compacted chromatin
in mammalian cells that is independent of DNA synthesis (Alexandrow &Hamlin,
2005; reviewed in Alabert &Groth, 2012). The mechanism of Cdc45-induced
chromatin decondensation is unclear but seems to involve recruitment of S phase
34
kinase cyclin A-CDK2 and phosphorylation of linker histone H1 synthesis
(Alexandrow & Hamlin, 2005).
Structure of CMG complex
Single-particle EM reconstructions comparing Mcm2-7 and CMG complex provides
structural insights into mechanism of helicase activation (Costa et al. 2011). As
previously mentioned, free Mcm2-7 exists in both an open, "lock-washer" form and
a closed "notched" form (Figure 6). Binding of Cdc45 and GINS seems to stabilize the
notched form of Mcm2-7 and seal the Mcm2-5 gap. The closure of Mcm2-5 gap is
further stimulated by ATP binding, (Costa et al. 2011), the resulting "locked" form
of CMG has a central channel within the Mcm2-7 ring and a side channel between
external face of Mcm2-7 and GINS-Cdc45 (Figure 10). Interestingly, this structural
data correlates well with the previous biochemical finding of an Mcm2/5 "gate" that
closes when Mcm2/5 binds ATP (Bochman & Schwacha, 2007, 2008). The closed
conformation of Mcm2-7 could be critical for efficient ATPase and DNA unwinding
activity. In vitro studies demonstrated that CMG complex has a higher affinity to
DNA substrates than Mcm2-7 alone. Moreover, CMG demonstrates significantly
higher helicase and ATPase activity but similar ATP binding rate, suggesting Cdc45
and GINS induce an Mcm2-7 conformational change that alters ATPase active sites
(Ilves et al. 2010). Based on both the structural and biochemical evidence, it is
possible Cdc45 and GINS stabilize the closure of the Mcm2/5 gate, which activates
Mcm2-7 ATPase activity or prevent Mcm2-7 dissociation from the DNA.
35
Alternatively, GINS and Cdc45 might improve helicase activity by facilitating DNA
capture by Mcm2-7 or they might act as a scaffold to hold the displaced ssDNA.
Figure 10
A
Mcm2-7
Cdc45
Notched
ATP
Mcm2-7
GINS
B
Mcm2-7
tGINS
Notched
CMG
Locked
CMG
Lock-washer
McmT2-7
GINS
eMcm2 o Mcm6 o Mcm4 0 Mcm7 0 Mcm3 0 Mcn5
Figure 10. Closure of Mcm2/5 gate is stimulated by binding of helicase
activators and in presence of nucleotide. Left. Single-particle electron microscopy
reconstructions of DrosophilaCMG showing Cdc45 and GINS bridge the Mcm2/5
gate (A). Addition of ATP analogs narrows the gap of Mcm2/5 (B). Right Model that
ATP, Cdc45 and GINS together results in closure of Mcm2/5 and presumably lock
CMG into an active form. (Figure adapted from Costa et al. 2010)
36
Thesis Summary
In this study, I dissected the function of yeast Cdc4S using a combination of in vivo
and in vitro approaches to elucidate the mechanism of Mcm2-7 helicase activation.
To map the functional domains of Cdc45, I generated a series of deletion and sitespecific Cdc45 mutations based on homology, structural prediction algorithms and
sequence conservation. Using in vivo complementation, I isolated three temperature
sensitive and five lethal site-specific mutations, and showed the N-terminus
(including the conserved motifs in RecJ homogloy region) and C-terminus of Cdc45
are both essential for its function. Using bioinformatics tool, I predicted a conserved
intrinsically disordered region (amino acids 169-209) which is dispensable for
Cdc45 function in vivo but site-specific mutations within the IDR region are lethal.
Mutants that failed to fully complement a CDC45 deletion in vivo were purified and
tested for their ability to complement Cdc45 function biochemically. Purified Cdc4s
mutants were found to be associated with Hsp70 chaperones, and subsequently
failed to fully restore in vitro replication in a Cdc45-dependent biochemical assay.
Co-immunoprecipitation studies of Cdc45 revealed an interaction with Sld3 and
Sld7. Using this assay, I mapped a region of Cdc45 (amino acids 208-303) that might
be required for Sld7 binding. Together, these preliminary data provide a starting
point for future mechanistic studies to understand role of Cdc45 in helicase
activation.
37
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47
Chapter II
Role of the S. cerevisiae helicase
activator protein Cdc4S
48
Summary
The mechanism of helicase activation at molecular level is not well understood.
Although a number of proteins are required for helicase activation, how they
function during this event is unclear. The Cdc45 helicase activator is a particularly
interesting protein that is required for these events. Cdc45 is recruited to
replication origin in late G1, directly interacts with Mcm2-7 and is required for
ongoing helicase activity. In this study, I examine the function of Cdc45 in budding
yeast DNA replication by mutational analysis. Deletion analysis of Cdc45 revealed
that both its N- and C-terminal segments are essential in vivo. Site-directed
mutations introduced at highly conserved residues, highly charged patches regions
and within a RecJ homology domain of Cdc45 resulted in the identification of five
lethal and three temperature-sensitive cdc45 mutants. I also identified a region of
conserved intrinsic disordered region (IDR) that is dispensable for viability in vivo,
but site-specific mutations of negative charged residues within the IDR region cause
lethality. Co-immunoprecipitation studies of Cdc45 revealed interaction with Sld3
and Sld7, and mapped a Cdc45 region (amino acids 208-303) that appears to
interfere with the Sld7 interaction. My work provides a starting point for
understanding the role of Cdc45 during helicase activation.
49
Introduction
Eukaryotic DNA replication initiation is a tightly-regulated, multi-step event that is
temporally separated into helicase loading and helicase activation to ensure that the
genome is duplicated exactly once per cell cycle (reviewed in Labib, 2010; Bell &
Dutta, 2002). During G1 phase, the Mcm2-7 helicase is loaded as a head-to-head
double-hexamer topologically linked with double-stranded origin DNA (Evrin et al.
2009; Remus et al. 2009; Gambus et al. 2011). This process requires the helicase
loading factors Cdc6, Cdtl and the origin recognition complex (ORC) (Bell &
Stillman, 1992; Takara & Bell, 2011; Frigola et al. 2013; Chen S et al. 2007), The
loaded Mcm2-7 double hexamer remains catalytically inactive until entry into S
phase, when it is activated for DNA unwinding and incorporated into bi-directional
pair of replication forks (Gambus et al. 2006; Tercero et al. 2000).
A key event during helicase activation is the association of two helicase activators,
Cdc45 and the GINS complex, with the loaded Mcm2-7 to form an active Cdc45-
Mcm2-7-GINS (CMG) complex (Moyer et al. 2006; Ilves et al. 2010). This event is
triggered by an increase in S-CDK and DDK activity during G1 to S transition. The
origin recruitment of Cdc45 is dependent on DDK activity while recruitment of GINS
is dependent on the additional activation of S-CDK activity (Heller et al 2011).
Multiple replication initiation proteins participate in the stepwise assembly of CMG.
In budding yeast, DDK targets loaded Mcm2-7 subunits, primarily Mcm 4 and Mcm6
(Randell et al. 2010; Francis et al. 2009). DDK phosphorylation is required to
50
alleviate an inhibitory function of Mcm4 N-terminal domain (Sheu & Stillman, 2006;
2010) and drive origin association of Sld3-Sld7 and Cdc45 (Kamimura et al. 2001;
Heller et al. 2011; Tanaka S. et al. 2011). Interestingly, although DDK is typically
thought of as acting in S phase, Sld3, Sld7 and Cdc45 are recruited to a subset of
early-initiating origins of replication during G1 in a DDK-dependent manner
(Aparicio & Bell, 1999; Heller et al. 2011, Tanaka S et al. 2011). Entry into S phase is
associated with activation of S-CDKs that phosphorylate Sld2 and Sld3 (Tanaka S. et
al. 2007; Zegerman & Diffley, 2007). Phospho-Sld2 and phospho-Sld3 bind Dpb11
and these interactions are required for the association of GINS and the leading
strand DNA polymerase (DNA Pol epsilon) with replication origins (reviewed in
Araki H, 2010; Sengupta S et al. 2013; Muramatsu S et al. 2010). Once assembled, the
CMG complex functions as the replicative helicase by acting as a ssDNA translocase.
Each CMG complex contains a single Mcm2-7 hexamer and moves in a 3' to 5'
direction along the leading strand DNA template (Ilves et al. 2010; Pacek et al. 2006;
Gambus et al. 2006, Fu et al. 2011). Consistent with the notion that CMG acts as or as
part of the "unwindosome", CMG is required for both establishment and
maintenance of replication forks (Gambus et al. 2006, Tercero et al. 2000; Kanemaki
et al. 2003).
Many mechanistic details of helicase activation are yet to be unraveled. One key
factor that is required for helicase activation and function is the essential helicase
activator Cdc45. Cdc45 is recruited to replication origins during late G1 to S phase in
a DDK- and Sld3-dependent manner (Heller et al. 2011; Kamimura et al. 2001).
51
Cdc45, in conjunction with the GINS complex, directly activates Mcm2-7. Cdc45
physically interacts with Mcm2-7, Sld3 and GINS, and possibly ssDNA (Moyer et al.
2006; Bruck & Kaplan 2011, 2013; Krastanova et al. 2012). However, the
significance of these interactions for helicase activation and how they activate
Mcm2-7 function is not well-defined. In addition, the domains of Cdc4s that are
responsible for these interactions remain unknown.
Bioinformatic analysis reveals that Cdc45 is a related to the DHH (Asp-His-His)
family phosphoesterases that includes the bacterial ssDNA-specific exonuclease
RecJ (Sanchez-Pulido et al. 2011; Krastanova et al. 2012 ; Makarova et al. 2012).
DHH family phosphoesterases are defined by four conserved N-terminal motifs
(Motif I to IV) that are essential for catalytic activity. Close inspection of the Cdc45
amino-acid sequence reveals several alterations in the N-terminal consensus
sequences that would be expected to inactivate exonuclease activity (Kratanova et
al. 2012). In agreement with this notion, biochemical evidence from human
recombinant Cdc45 revealed that Cdc45 does not have any nuclease activity, even
though it retains ssDNA-binding activity (Kratanova et al. 2012). Nonetheless, the
evolutionary link between Cdc45 and RecJ could shed light on the structure and
function of Cdc45.
The budding yeast S. cerevisiae has proven to be an excellent model organism for
mechanistic studies of DNA replication initiation. Unlike typical eukaryotes that
have poorly-defined replication origins, budding yeast has short, well-defined
52
autonomously replicating sequences (ARSs) that act as replication origins (reviewed
in Bell & Dutta, 2002). Yeast DNA replication has long been studied by molecular
genetics and biochemical techniques, leading to the discovery and characterization
of many replication factors that are conserved in other eukaryotes (reviewed in
Remus & Diffley, 2009; Sclafani & Holzen, 2007).
In this study, I dissected the function of budding yeast Cdc4s using a combination of
in vivo and in vitro approaches. Using a series of Cdc45 deletion mutants, I show that
both the N- and C-terminus of Cdc45 is required for its essential function. I also
describe new Cdc45 mutants that can be subjected to further biochemical and
genetic characterization. Intriguingly, I identified a putative IDR region that is
dispensable in vivo, yet site-specific mutations within this region are lethal.
Mutations in RecJ motifs of Cdc45 are either lethal or temperature-sensitive,
suggesting the RecJ homology plays important role in Cdc45 function. Finally, I
mapped a Cdc45 region (amino acids 208-303) that could be important for Sld7
interaction. My findings provide new tools to study Cdc45 and the mechanism of
eukaryotic helicase activation.
53
Results
Mutagenesis of Cdc45
To identify functional domains within ScCdc45, I constructed a total of ten CDC45
deletion mutants from N- and C-terminal ends (AN1-100, AN1-197, A1-303, AN1438, A1-590; AC597-650, AC448-650, A311-650, AC204-650, AC109-650). In the
hope of maintaining Cdc45 protein folding, the end points for the truncations were
determined based on a secondary structure prediction tool (Jpred3,
http://www.compbio.dundee.ac.uk/jpred, Cole et al. 2008).
I also applied a computational tool that predicts site of disordered protein structure
(PONDR VL-XT, http://www.pondr.com, Garner et al. 1999; Tompa &Fersht 2010)
to Cdc45. This program identified a statistically highly significant intrinsically
disordered region (IDR) between residues 169 to 229 of S. cerevisiaeCdc45 protein
(PONDR score = 0.9460, Figure 1). This 61-amino-acid predicted IDR contains highly
acidic patches and 20 amino acids that function as a nuclear localization sequence
(NLS) (Hopwood and Dalton, 1996, Pulliam et al, 2009). The presence of the NLS
suggests that the IDR is likely surface-exposed and accessible. Interestingly, the
PONDR-predicted IDR region is well conserved in Cdc45 from yeast to human. To
evaluate whether this conserved IDR is important for Cdc45's function, we also
generated an internal deletion of the IDR while leaving the NLS intact.
As an alternative approach to identify residues that contribute to essential Cdc45
function, we introduced alanine substitutions at sites of highly conserved or clusters
54
Figure 1
CdO45
Generated at pondr.om, th: Tue Mr 27 01:42:11 2012
1.0-
0.8-
A0.6-
c 0.50.4-
0.2
0.0-,
V.-
100
200
300
400
600
ResIdue Number
GDDELSGDENDNNGGDDEATDADEVTDEDE
EDETISN KRGNSSIGPNDLSKRKQKQRKKQ
D
Figure 1. Identification of a conserved intrinsically disordered region in
budding yeast Cdc45 using PONDR-VL-XT. The cutoff PONDR score on the y-axis
for a protein domain to be considered as "disordered" is 0.5. The highly significant
disordered region (IDR) is predicted to be a.a.169 to 229 (Average strength/PONDR
score = 0.9460), where the residues are bolded. Sequence of the IDR region is also
shown. Blue: NLS sequence. Red: two negatively charged patches (171-173; 199201) that when mutated to alanine were lethal.
55
of charged residues in Cdc45. Alanine substitution removes side-chain functional
group of the targeted amino acids, but should retain normal backbone conformation.
I chose conserved residues based on multiple sequence alignment of Cdc45 genes
ranging from yeast to human (Figure 2). Patches of charged residues were targeted
when three charged residues were found out of four consecutive amino acids. These
charged patches are more likely to be found on the protein surface and often
participate in protein-protein interactions (Cunningham &Wells, 1989).
Figure 2
36-37 C40A
Drosophila
Human
Xenopus
Budding
Fission
NFV--QDLRNDFYROL---VGIR-ILIVVNYDIDICASRILOALFYDHMLYTVVP
------ QSQR-VLLFVASDVDALCACKILQALQCDVQYTLVP
MFV--SDFRFY
NFV--SDLRKEFFDVI------VTER-VLLLVAPDVDALCACKILQALFCDHVQIYTLVP
CITIOELS-LFKKQLVQSQIVP
NYYGISQYSIAYNKILRNSSSESSCQLVIFV
HFIKRSDYABAYLXKIA-SVSGGCTVQLPV-ALDPDALCACKLLSTLLKGDFISHEIRP
a *
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3 **:** ::*. :3 a :
. :
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.3
66-69
Drosophila
Human
Xenopus
Budding
Fission
D91A
INGVTGLHAMYGI---QGDVKYVVLVNCGGCVDVIELLQP----------------SDD
VSGQILITAFLI---KIQFUYFILNCGRVDLLDILQP----------------DED
VSGQILITLFLI---KIQfRYFVLINCGANIDLLZTLOP---------------- QI
IFGYS W SQL---DDN SLLLVGFIDPQIVIDTDEKSGQ8FR
VSGYRDLBQANKTLLIQNEDIXFIILLNCGTNVDLNYLVS--------N--------ED
0
S**
.:*
*
:*3
3*
*
Human
Xenopus
Budding
Fission
71473
154-57
124-126
Drosophila
VTFFICDflRPLDVCNIYSDRQVCILGDSLIENPAFTIFYDBIGIDIDIDISSDTIQ
TIFFVCDTURPVNvnVYnDTQILLIKQDDDLVPAYIZFD-IDU--BGNDSDGSI
AIFYICDTURPIDVVWITNDSQVXLLIRQDDDLIPAYDDIFNDDIIDGDSG1SDGAB
DLSGD
RDIVVZP WHLAIXFGSQIIC
VSIYVIDSURPIDLUNIYIUNZIFVFDDGDIIIODIODANTAFNSNILS-------DS
333
*3***
3
3303 *: .
3
3
.
8
ltO-1,3 19-201
Drosophila
Human
lenopus
Budding
Fission
QNDDSGA---------------ZSDQIDQAPRSRILSRLIRUIQRILQRARRQWBS
SKRTRLIIIIVO-NRRRORRZNVA
------------------------------flRFDAVERRIWIQtE=IlA
P-------SG
------------------------GSZIGPD-LS iCQRKIQI
IM,001----DKTIS
UDNGGD
SYSSDYQARSRRRFSITTOR-RAZIKIRRKRU
INSDSSNER---- UZYMUM
Drosophila
Human
Xenopus
Budding
Fission
IJIJWFZIkLANKIAIDNNDLafr1?QLLL1IrNAT
IRDRIX-FVITOFSYYGRSA
RNlDIL-FDNBOYITGTBBANV FILMUIL8XDLND LWTAVGLTDQWYQDQITG6KY
LAWnNSDSNDNLAVGLTDQWYQDPITLNY
RR3UII-FDYQYIYBGBSANfl
LG'TSLDIAY---AQVY
AMOMTTWSXISAQIYSANUGELBMML
yIFASILSTYIKGSWYGESITNIFAVASNLGRDNDNLLAIVGLTCLBIICOSSXTF
I
*
:
** **
3..
* I is :
. : * I
238-240
56
297-300
D314A
336-337
Drosophila
Human
Xenopus
Budding
Fission
TLrLBQIQSHVSRLTNKTND-----WN---TNSASKITENDLLVLYRBWPVTSNRYS
VTDVGVIRVSREINHNED-----ENTL8VDCTISFYDLRLVLYQBWWLHSLCUT
Drosophila
Human
xenopus
Budding
Fission
RYSBCLVHAAQTGANDLVLRRFFSKVULAKYDI
DVGTLORBVSRENBGaED-----USLSIDCNRIAFYDLRLSLYHVSLYZSICNS
MLYP
TPSSR ------N
TIPDYYLFLLRRSSLM
S
NRSYSLLKD3VNRLUPSPLENOIVGRAAGTPHDQSIR*DEFRNLMVRUSLYDSNLNS
36-366
386-387
SYTAABFKLWSVUGQRLQZLADNGLPLIQWQKFQAMDISLUCNLRKNXEQSAUKFGI
CYTSATLL
GDISLKBNLRENLEESAVKFGH
IIFDMDu
YGL
LIMUSSLMAPTYG
MTVHAKLSLKARNOIPLSTAQ3TVLYNDHUl
AYGSRLB
*
G
L
G
C
I
. S: *. .Slot::
:
of:
: :St:. t
.
: t
457-460
Drosophila
Human
Xenopus
Budding
Fission
ADIVYGTFTLSYGYRSRYAAADYVYALLAINESVKH----------------------KDRVQTFSIHJGFKHKFL&SDWFATNSLKNSPZKD----------------------KDVRVQT7SVQ7G7KNKFLADIVFAVLSLL3NTRRD----------------------QDIIRDGFVRTLGYRGSISASEFVALTALLVGSTDxDSvKINNDDT
DDVFSFTRTYGFCTLSASDVSYAISALL
GNTGvLLQsRTvARSPDmT----ZBY
0**:
I
*tt
*
::* a
Drosophila
Human
Xenopus
---------
470-472
Budding
Fission
48548
--------- GSGTDHFIQALDLSRNLDLYGLELAKKQLRAT2QTIASCLCTNLVIS
--------MGTDNFIKALDSLSRSNLDXL
E
CIQQTVASCICTNLILS
ALT-TGILOLAITGA
Drosophila
Human
Xenopus
Budding
Fission
E
LzRFEMAoNozvLmnpYDALDD--VDSLERALLAMLQRAIVRTGITLLZKRAIKT
*
Drosophila
HUan
Xenopus
Budding
Fission
515-517
KTPEDCFLEADAGIDOAKLLBAAVFQVQSSLZARQVBS
a:':.
:
.:.
*
.
.
:
*.
658-561
636-37
AGSFFYYVLQZ---BAFSYPYALGLLARFLLRGVATSRA-RQASDLPLIASCPLNAS
QGPFLYCSGTPDVNLFSRPALSLLSLLKSNVCTK-RRCKLLPLVMAAPLSB
QGPFLYCYIJEGTPDVMIFSNPISLCLLCKYLLKSiVCSTN-KRCKLLPLVLAAPLDA
LRIYRLCVLODGLYRNPLTLLRLGNLICC
---- KLLPVLAS-IDEN
LRSFRFGLINEG-PDIIFNPLALTKSLVIAEAINEQUBRGKLRHLPLVLAA-Fvu
BGCLLVGIVPVR---------DS-PRNFFGAFEQAAOISGVALLQDFFIPAVVQLRQ
BGTVTVVGIPPET---------DSSDRKNFFGRAPEIAAZSTSSRNLNHDLSVIZLKA
KGTDIVGXPPEA---------ZSSDINFFGRAFKAAESTSSRTLHNHFDSIIZLRT
TDTYLVAGLTP-RYPRGLDTITKKPILNHFSNAPQITATDAMvRIDFEsSIII
KHRYL VA
ANNTSATL0DCASVImCQ
a.*.
* '.
** "a: a a
: '*a ::: :
4636
Drosophila
Ruman
Zenopus
Budding
Fission
SDLTRFLDSLTVLLABDRSKFLDMLISLLSZDRSKILDALISLLSaSjpLCTLSGLL
SDOVFLSLSFKLL
.*
U
N
*
**S *
RWcJ Homology
COMrVed Mek
A
s
n
Figure 2. Site-directed mutagenesis of Cdc45. The multiple sequence alignment
of Cdc45 homologs (Cdc45 protein sequences of Drosophila, Xenopus, human,
budding yeast and fission yeast) was performed by Clustal Omega
(http://www.ebi.ac.uk/Tools/msa/clustalo/). ScCdc45 sites that were targeted for
mutagenesis in this study were labeled and highlighted.
57
Cdc45 shares homology with DHH family pyrophosphatases and exonucleases
including Reci (Sanchez-Pulido et al. 2011, Krastanova et al. 2012). To examine
importance of Cdc45 regions that show homology to RecJ, I used a protein homology
detection structure prediction tool (HHpred, Soding et al. 2005,
http://toolkit.tuebingen.mpg.de/hhpred) that employs structure-based profile-toprofile alignment and found a RecJ homology region at N-terminus of Cdc45.
Unlike other studies that indicated RecJ homology extends throughout the Cdc45
sequence (Krastanova et al. 2012), HHpred analysis in this study found residues 8128 of S. cerevisiaeCdc45 that were related to the N-terminal catalytic domains
(Domain I and part of domain II) of T. thermophilusRecJ (E-value = 0.057)(Figure 3).
This N-terminal RecJ homology region of Cdc45 will be called DHH domain in the
remainder of the chapter, as it contains conserved catalytic motifs I to III that are
shared by all members in the DHH family.
To determine whether the putative N-terminal RecJ homology is important for
Cdc45 function, I eliminated key regions of similarity between Cdc45 and RecJ by
mutating conserved catalytic motifs that are predicted to form part of the catalytic
center of Cdc45 DHH domain (Sanchez-Pulido &Ponting, 2011). The two sites I
targeted in ScCdc45 DHH domain are NID (a.a. 35-57), which is within Recj motif I,
and DAH (a.a. 124-126), which is in RecJ motif III (Figure 3).
58
Figure 3
Cdc45
-----
s FsnnEuM:ssssseQ:::Tisc!.D
Z:,TsiJg:::gsQ:,p-
- --
:TGYS;.MYSQDWESZ
.:
83
6O
NID vs DAD
S8
:DAnAA.AmAGea---::
cnzc----------------ccn
a-----uucs
-----
Note----
134 (666)
Red
motif 2
motif 1
Cdc45
9*ce-*e&MddgkCChNaccccecccccc*mtuccc
4
128 (6so)
$
13S '1G:1UHALM
---------------
sccc ------------------------gccccmmm
DAH vs DHH
T ::TDHTP
uu99ccc
--------------- xc*Z&c
163 (666)
Red
motif 3
Figure 3. HHpred profile-to-profile alignment of ScCdc45 and TtRecJ. Close
inspection of sequence similarity between ScCdc45 and TtRecJ based on HHpred
analysis, which identified significant similarity between amino acids 8-128 of
ScCdc45 and amino acids 58-163 (Domain I and part of domain II) in TtRecJ. It was
noted that the conserved RecJ motifs are mutated in ScCdc45. Two sites (NID and
DAH) within conserved motifs of ScCdc45 are chosen to be targeted for mutagenesis
in this study.
In vivo complementation analysis of Cdc45 mutants
I first tested all the Cdc45 deletion mutants for their ability to complement a CDC45
deletion in vivo (Figure 4). All the N- and C-terminal truncation mutants are nonviable either with (Figure 5) or without a C-terminal epitope tag (data not shown).
One caveat is that the lethality of the five of the truncation mutants (A1-303, A1-438,
A1-590, A204-650, A109-650) might simply due to defects in Cdc45 nuclear
localization, as these mutants lack the NLS sequence (a.a.209-228). Addition of the
SV-40 NLS to these deletion mutants would be required to address this possibility.
On the other hand, the smallest N-terminal truncation (A1-100) and C-terminal
59
Figure 4
In vivo complementation analysis
Cdc45 swapper strain:
Endogenous Cdc45
Acdc45
replaced by KanMX4
&cdc4S
ura3
KanMX4
ENrl4
U
Ieu2
+ 5-FOA
selection
Mutants failed to
complement or survive:
Purified for in vitro
replication assay
Figure 4. Schematic of the in vivo complementation screen for Cdc45 mutants.
A haploid yeast "swapper" strain was constructed that has deletion of the
chromosomal CDC45 gene. The strain survives due to the presence of the wild-type
CDC45 gene in a plasmid with a URA3 marker. The LEU2-marked plasmid constructs
containing various Cdc45 mutations were integrated into the leu2-1 locus of the
swapper strain. These cdc45 mutants were expressed under the control of the wildtype CDC45 promoter and 3'UTR. To assess complementation, the resulting strains
were grown in the absence or presence of 5-fluorootic acid (5-FOA), which selects
against the URA3 marked wild-type CDC45 plasmid. Mutants that grow on 5-FOA
retain the essential functions of Cdc45 whereas mutants that die on 5-FOA lack at
least one essential function.
60
Figure 5
1
Viabilt
128
169
229
6W0Vablt
-.
FL 1-650
V1
AN 1-100
X
A169-208
v
AN 1-197
X
AN 1-303
X
AN 1-438
X
AN 1-590
X
AC 597-650
X
X
AC 448-650
AC 311-650
K
AC 204-650
K
AC 109-650
X
3X HA 3X FLAG
DHH domain
IDR
M
NLS
Figure 5. In vivo complementation analysis of Cdc45 deletion mutants. The
diagram of full-length Cdc45 shows DHH domain (a.a. 8-128) that has RecJ
homology predicted by HHpred (green box) and a putative IDR region predicted by
PONDR VL-XT (a.a.169-229) including a NLS (a.a.209-229, orange box). The N- and
C-terminal truncation constructs were designed based on Jpred3 analysis, except
A169-208, which is an IDR (a.a.168-208, purple box) deletion mutant. All constructs
were C-terminally tagged with tripe HA and triple FLAG tag (yellow box) for protein
purification. The result of in vivo complementation are shown on the right (the
viability column), a green tick check mark indicates the deletion mutant is able to
complement loss of CDC45 function whereas a red cross means the mutant fails to
complement the CDC45 deletion.
61
truncation (A597-650) with intact NLS failed to complement a CDC45 deletion in
vivo, arguing both the N- and C-terminus of Cdc45 are essential for its function. Only
the integral IDR deletion mutant (A169-208) complemented the loss of CDC45,
indicating the IDR is dispensable for Cdc45 function (Figure 5).
Using the same in vivo complementation analysis described above, I asked if any of
the site-directed mutations are critical for Cdc45 function (Table 1). To identify both
fully inactivated and temperature-sensitive (ts) mutations I tested the mutants for
growth at both normal (250C) and elevated (370C) temperatures. I isolated five
lethal mutations (alanine substitution at positions 35-37; 66-69; 171-173; 199-201;
336-337) and three ts mutations (alanine substitution at positions 124-126; 238240; 485-488) (Figure 6A). The ts mutants were not sensitive to both low and high
dose of hydroxyurea (HU) (Figure 6B).
Out of the eight mutations, two lethal mutations (35-37; 66-69) and one ts (124126) were found in the N-terminal, RecJ DHH domain. These results indicate that the
N-terminal DHH domain, although likely catalytically inactive, is still essential for
yeast Cdc45 function. Surprisingly, two lethal mutations (171-173; 199-201) are
found in the IDR region that was dispensable when the region was deleted (Figure 5
& 6A). This finding suggests the mutations are dominant negative, but further
experiments would be required to confirm this hypothesis (e.g. monitoring the
effect of overexpression of this mutant in a wild-type strain).
62
Table 1
I Cdc45 site specific mutations
166-69 (ELRR -> ALAA)
I
I C40A (C->A)
154-157 (EQKE -> AQAA)
I
Viability in vivo
I
-
I
+
I
+
190-199(DADE->AAAA)
+
I
238-240(EEY->AAA)
I D314A(D->A)
356-358(KKR -> AAA)
ts
I
+
I
+
457-460( EEED->AEMA)
I
I
485-488( DDRK->MARA)
ts
515-517( EKKAKA)
558-561(ESED -> ASAA)
Table 1. Table of in vivo complementation results of Cdc45 site-specific
mutants. "+" indicates the mutant is able to complement loss of CDC45 function
(viable on 5-FOA); "-" means the mutant fails to complement the CDC45 deletion
(lethal on 5-FOA); "ts" indicates the mutant is viable at 250C, but fails to grow at
370C.
63
Figure 6
A
Ts and lethal site-specific mutants Identified:
485-488(DDRK->AARA)
238-240(EEY->AAA)
Or
124-126(DAH->AAA)
amaa
DHH
domain
aE t
IDR
484-488 DDRK->AAAA)
a
E E
Cdc45
650
336-337(DS->AA)
199-201(EED->AAA)
171-173(DDE->AAA)
a
66-69(ELRR->ALAA)
35-37(NID->AAA)
x
recJ homology
conserved residues
a alanine scanning
B
t=15'
t=4hrs
t=2hrs
t=8hrs
WT
124126
238240
460466
Figure 6A & B. In vivo phenotype of isolated Cdc45 site specific mutants. A.
Among the sites targeted for site-directed mutagensis, five lethal mutations (shown
in red) and three temperature-sensitive mutants (shown in blue) were isolated. The
locations of these mutations on the Cdc45 protein sequence and the changes that
were made are indicated. The color of the associated "X" indicates the strategy by
which the mutation was generated. B. The temperature sensitive mutants (124-126;
238-240 & 485-488) do not render sensitivity to 200mM HU (Mutant 238 displays a
poor growth at 30*C, independent of HU treatment). Cells were grown in YPD to O.D.
at 0.5 before adding a-factor for G1 arrest. Synchronized cells were washed to
remove the a-factor and released in YPD with 200mM HU at t=0. Serial dilution was
performed and aliquots of cells were plated on YPD at 30*C at different times.
64
Purification of Cdc45 mutant proteins
To characterize the defects that lead to the lethality of the Cdc45 mutants in vivo, I
attempted to biochemically purify the corresponding proteins for in vitro analysis. I
generated yeast strains that overexpressed wild-type and all of the Cdc45 mutants.
Each expression construct included a 3X HA and 3X FLAG epitope tag fused to the Cterminus of the Cdc45 mutant to facilitate immunoblot detection and protein
purification. Previous studies showed that wild-type Cdc45 protein overexpressed
and purified from yeast using the same epitope tag was functional (Heller et. al.
2011).
Initial studies to check the level of Cdc45 wild-type or mutant protein expression
after galactose induction revealed that most truncation mutants had similar or
partially reduced levels of protein expression compared to that of full-length Cdc45
(Figure 7). The only exception was the largest N-terminal truncation (AN1-590)
that expressed at much lower levels (just above the level detectable by
immunoblot). Three of the N-terminal site-specific mutations (35-37; 66-69; 171173) showed either consistently no observable expression (35-37), or "unstable"
protein expression levels (66-69; 171-173). The "unstable" protein expression of
66-69 and 171-173 is enigmatic, as analysis of different isolates of the same
sequence-verified mutation gave rise to distinct results. In the cases of both 66-69
and 171-173, with confirmed Cdc45 site-specific mutation there are yeast isolates
that showed no observable overexpression (e.g. 66-69 in Figure 7), or overepression
levels like wild-type (e.g. 171-173 in Figure 7). These inconsistent results might be
65
Figure 7
FLCdc45
+
AC 597-650 AC 448-50 AC 311450 AC 204-50 aC 109450
AN 1-100 AN 1-197 AN 1-303 AN 1-436 AN 1-590
-+
+
+
+
-4.
4.-4.
-4.4.-4.
all
12435-37 66-69 126
171173
199- 238201 240
336337
"ni
A
485488
4
4
4
- 4
-. +
-
.+
-4.
-4.
-4.
.4.
- : Glucose induction
+ : Galactose induction
Figure 7. Cdc45 mutant protein overexpression under galactose induction. All
Cdc45 mutant (HA-tagged) overexpression strains were grew in YPGlycerol, before
induction of overexpression by adding galactose to the growth media. At
approximately O.D. = 0.8, cells were isolated and subjected to TCA precipitation. The
resulting protein samples were run on 8% SDS-PAGE and immunoblotted with antiHA. Glucose was used as a negative control to make sure the protein expression of
Cdc45 mutants was specifically induced by galactose.
66
due to secondary mutations that arose within or outside the Cdc45 gene in yeast
genome. Preliminary sequence analysis of the Cdc45 gene PCR amplified from
genomic DNA did not reveal secondary mutations.
Many of the mutant Cdc45 proteins co-purified with Hsp70. Wild-type Cdc45 after
purification was predominantly a single band of the appropriate size. However,
using the same purification approach, I observed several unknown bands copurified with all the N- and C- terminal truncation mutants (Figure 8A). Mass
spectrometric (MS) analysis of the eluted mutant proteins identified large amount of
the Ssa family of Hsp70 heat shock proteins. In addition, Hsp70 co- chaperone
proteins were also identified (Table 2). Consistent with MS result, the extra copurified proteins migrate at approximately 70kDa, the predicted size of Hsp70. Copurification of these Hsp70 chaperones suggested that the truncations affect Cdc45
protein folding. Interestingly, Hsp70 contamination was not observed in
purification of the IDR deletion mutant, which is the only deletion mutant that is
able to complement in vivo (Figure 8B). The predicted Hsp70 contaminants were
also found in the FLAG purifications of many of the site-specific Cdc4S mutants
(124-126; 199-201; 238-240; 336-337; 485-488) (Figure 8B). Thus, although sitespecific mutations might be expected to preserve the overall protein fold, the nonfunctional alleles may still have a folding defect.
67
Figure 8
A
kDa
IAN1-100
82-
N-terminaltruncations
kDa
C-terminaltruncations
F
AN1-303 AN1-438 AC597-650 AC448-650 AC311-650
AN1-197
oft
~
~
-
-t
_
-82
-64
-49
FL
Cdc45
-37
B
kDa
336337
238240
199201
124126
485488
WT
AIDR
82-
0010.
64-
Figure 8. Purification of Cdc45 mutants found to be associated with Hsp70.
Anti-FLAG affinity purification of Cdc45 full-length and Cdc45 mutants were carried
out, followed by SDS-PAGE analysis with Coomassie blue staining. Cdc45 protein
bands were indicated by the red arrow (confirmed by anti-FLAG or anti-HA
immunoblot). A consistent set of extra bands at size around 70kDa were co-purified
with either Cdc45 truncation mutants (A) or site-specific mutants (B). The only
exceptions are purification of wild-type and Cdc45 IDR deletion (B).
68
Table 2
SSA1
Hsp70 chaperone; for protein folding
132
87.5%
and NLS-directed nuclear transport
SSA2
HSP70 family member, for protein
folding
119
82%
STI1
HSP90 co-chaperone, interacts with
91
72.8%
Ssa group of Hsp70 chaperones
HSC82
Hsp90 chaperones
104
69.8%
HSP60
Chaperonin to prevent aggregation
81
67%
and mediates protein refolding
YDJ1
Chaperone in regulation of HSP90
and HSP70 function
39
61.4%
RPS3
Protein component of 40S ribosomal
subunit
13
54.2%
SSC1
HSP70 family member
34
50.5%
SSB1/
SSB2
HSP70 family member
30
49.3%
SIS1
HSP70 co-chaperone, interacts with
Ssalp
21
48.6%
CDC45
Replication initiation protein
49
46%
Table 2. Summary of mass spectrometric analysis on a protein sample of
immunoprecipitated the Cdc45 mutant AC448-650. Besides Cdc4S, abundant
Hsp70 chaperones from Ssa/b family (SSA1 & SSA2, in particular) were identified,
along with co-chaperones. It was noted that ribosomal subunits were frequent
contaminants in purified protein samples.
69
Co-immunoprecipitation of Cdc45, SId3 and Sld7
Based on previous findings, Cdc45 forms a complex with Sld3 and Sld7 prior to
association with the Mcm2-7 complexes at the replication origin (Tanaka S. et al.
2011; Tanaka T. et al. 2011). It is possible that Cdc45 might be stabilized by
association with Sld3 and Sld7 and therefore prevents association of Hsp70
chaperones with Cdc45 mutants. To address this, I generated yeast strains that
overexpress either full-length or truncated FLAG-tagged Cdc45, as well as SId3 and
Sld7. Both Sld3 and S1d7 included a VS epitope tag fused to their C-terminus.
Galactose-induced overexpression of Sld3 and Sld7 was verified by TCA
precipitation and immunoblotting (Figure 9A).
Similar to a Cdc45 protein purification protocol, FLAG-tagged wild-type Cdc45 was
immunoprecipitated from yeast cell lysate using anti-FLAG agarose beads. Elution
fractions of FLAG-Cdc45 bound to beads were resolved on SDS-PAGE, followed by
immunoblot against VS antibody. The presence of the S1d3 and S1d7 bands on
immunoblot suggest that S1d3 and S1d7 could be co-immunoprecipitated with wildtype Cdc45 (Figure 9A), consistent with the previous published studies (Tanaka T. et
al. 2011; Tanaka S. et al. 2011). However, my co-immunoprecipitation (co-IP) study
lacked important controls such as analysis of a sample of the starting lysate, or
mock-IP samples (IP without anti-FLAG beads or using resin of an irrelevant
antibody) to show that the binding of Sld3 and S1d7 to FLAG-Cdc4s was specific.
Prior to following up on these studies these controls should be performed.
70
Figure 9
A
B
82-
82-W
26-
kDa
C
1
2
1
2
Anti-V5
a-Cdc45
kDa E2 TCA
Figure 9. Sld3 and SId7 co-immunoprecipitated (IPed) with wild type Cdc45. A.
Protein samples prepared from E2 fraction of FLAG IP (left) or whole cell TCA
precipitation (right) were analyzed by 12% SDS-PAGE and anti-V5 immunoblot (for
Sld3/upper band and Sld7/lower band). B. Coomassie staining of E2 fraction from
FLAG IP in presence of overexpressed Sld3 and Sld7 showing FLAG-Cdc45. C.
Immunoblot analysis of wild-type Cdc4S IP from E2 fractions overexpressing Cdc45,
Sld3 and Sld7. The amount of Cdc45 in the protein samples was controlled. Lane 1:
both Sld3 and Sld7 were V5 tagged. Lane 2: Only Sld3 was V5 tagged, while Sld7 was
untagged.
71
The expected size of co-purified Sld3 is approximately 80kDa on SDS-PAGE, similar
to that Cdc45 (Figure 9B). This co-migration of Sld3 and Cdc45 made it hard to
distinguish between the two proteins by non-specific protein staining. On the other
hand, the predicted molecular weight of Sld7 is approximately 30kDa. Importantly,
VS-cross-reacting band of this size was lost when Sld7 was untagged (Figure 9C,
lane 2), this identified the 30kDa band in this co-IP study to be Sld7. However, the
amount of Sld7 co-purified was too low to be detected by Coomassie staining
(Figure 9B).
The Cdc45 FLAG-IP experiments were repeated with five different Cdc45 deletion
mutants (AIDR 168-208, AN1-100, AN1-197, AN1-303, AC448-650 and AC311-650).
Sld7 could be immunoprecipitated with all of the Cdc45 deletion mutants except
AN1-303 (Figure 10A & C). The lack of co-IP of Sld7 in the case of Cdc45 AN1-303
mutant indicates that the co-IP of Sld7 requires Cdc45 and not due to cross-reaction
with the FLAG antibody. Since Sld7 bands were still present in AN1-197 and AIDR
168-208, the result suggests that region between amino acids 208-303 of Cdc45 is
important for Sld7 interaction.
Based on the result of overexpression analysis (Figure 9A), it is likely the upper
band detected by anti-V5 immunoblot at about 80kDa is V5-tagged Sld3 but caution
must be taken to verify the band by repeating the experiment with Sld3 fused to an
alternative epitope tag. For wild-type Cdc45 and AN1-100, AN1-197 and AC311-650
72
Figure 10
A
AN1-100
B
AIDR
kDa
Coomassie Stain
82-
*
Anti-V5
6449-
E2
kDa
E3
E4
E2
AN1-197
E3
E2
E4
E3
E4
E2
AC448-650
AN1-303
E3
E4
AC311-650
AN1-100 AIDR
a-Cdc45
C
AN1-197
kDa
E2
E3
E4
AN1-303
E2
E3
AC448-650
E4
E2
E3
E4
AC311-650
E2
E3
E4
82-
-SId?
26-
-SId7
Anti-V5
Figure 10. Co-immunoprecipitation analysis with Cdc45 deletion mutants. IP of
Cdc45 truncation mutants in the presence of overexpressed Sld3 and Sld7, followed
by 12% SDS-PAGE and immunoblot analysis. A. The anti-Cdc45 immunoblot showed
the amount of Cdc45 input and the anti-V5 immunblot revealed co-purified Vstagged proteins. In case of Cdc45 AN1-100, the VS cross-reacting protein pattern is
similar to wild-type (upper band predicted to be Sld3 and the lower band Sld7,
compare to Figure 8A, lane of E2 fraction). In the case of Cdc45 AIDR 168-208,
instead of a single band at 82kDa, there were two differentially migrated bands
(indicated by red asterisks). B. 12% SDS-PAGE/Coomassie staining of the protein
fractions (E2 to E4) from Cdc45 deletion mutant IPs. The red arrow indicates the
expected size of full length Sld3 (approximately 8OkDa). The Sld3 and Sld7 proteins
seen for the Cdc45 deletion mutants have not been verified. C. shows anti-V5
immunoblot of the same fractions from the IP of Cdc45 mutants in presence of
overexpressed Sld3 and Sld7. The unknown bands are indicated by "T', but may
represent products of Sld3 proteolysis.
73
mutants Sld3 migrated as a single band at around 80kDa (Figure 10 A & C). In
contrast, the Cdc45 AN1-303 and AC448-650 deletion mutants, showed two
additional major bands below the predicted 80kDa Sld3 (one at -70kDa, the other
at -55kDa, Figure 10C). The lower band at around 55kDa was particularly intense
in AN1-303, which was also seen by Coomassie staining (Figure 10B).
Intriguingly, the 80kDa band expected to be Sld3 split into two bands in the IDR
deletion mutant (A168-208): one band migrates slightly slower while the other
migrated slightly faster (Figure 10A). A potential pitfall to drawing conclusions
based on the co-IP study could be that high level of protein expression of Sld3, Sld7
and Cdc45 might drive them to assemble into a non-physiological complex.
In vitro Cdc45-dependent replication assay
To gain further insights into Cdc45 function, we used in vitro biochemical assays to
characterize the molecular defects underlying Cdc45 mutations identified in vivo.
First, to establish a biochemical complementation system, we have modified the in
vitro origin-dependent, replication initiation assay described previously (Heller et
al. 2011). This assay recapitulates all of the events of replication initiation and
elongation (Figure 11A). To make the replication assay dependent upon the addition
of exogenous Cdc45, I have reconstructed the yeast strain used to make S-phase cell
extracts so that it does not over-express Cdc45. In the absence of Cdc45
overexpression, these extracts are unable to support replication initiation in vitro.
74
Figure 11
A
B
DDK
GI Arrested
Cell Extract
+ + - +
Cdc6 +
Oenagec
Origin
+ +
-
DNA
I
+ Purified WT or
mnutant Cdc4S
-
Cdc45 + ++
Form pre-RCs
Purified DDK
+
cdc7-4 ts,
S-phase arrested cell extract*
*d
d&
b
E only)
Anti-MCM
Anti-ORC
E
Test for the presence of replication products and
DNA bound proteins
Figure 11. In vitro Cdc45-dependent replication assay. (A) Assay was performed
using 125 fmol of 7.6kb ARS1 origin DNA template that were coupled to magnetic
beads. The DNA beads were incubated with Gi-phase cell extracts to allow helicase
loading, followed by addition of purified DDK and S-phase extracts that lack
overexpression of Cdc45. (B) The addition of lpmol purified Cdc45 is a determinant
for replication to occur (-Cdc45 control). The Cdc45-dependent replication assay is
also dependent on prior helicase loading (-Cdc6 control) and DDK activity (-DDK
control). The replication products were analyzed by alkaline gel electrophoresis,
followed by autoradigram. The chromatin bound Mcm2-7 and ORC could be
detected by immunoblot analysis.
75
Importantly, I have shown that addition of purified full-length Cdc45 to S phase
extracts prepared from cells lacking Cdc45 overexpression restores DNA replication
initiation and elongation. This Cdc45 dependent in vitro replication assay is also
dependent on prior helicase loading and DDK activity (Figure 11B).
I tested whether the Cdc45 mutant proteins complemented the Cdc45-dependent
replication assay. Since I focused on mutants that failed to complement a CDC45
deletion in vivo, I expected that most, if not all, purified mutant proteins would fail in
this replication assay. Nevertheless, I could investigate which steps of replication
are defective for each of the Cdc45 mutants by analyzing the proteins associated
with the DNA template in the presence of the mutant protein. For instance, Cdc45
origin association step can be assessed by the detection of DNA-bound Cdc45
mutants. If Cdc45 mutants are associated with origin DNA, I can further assess the
origin recruitment of downstream replication initiation proteins that require proper
Cdc45 function. As a result, helicase activation steps can be readily assayed in this in
vitro replication assay by immunoblotting for origin-associated proteins. In the case
of DNA unwinding, the association of the eukaryotic ssDNA binding protein RPA can
be monitored.
Preliminary results indicate that all purified Hsp70-bound, N- and C-terminal
truncated proteins failed to support in vitro replication. The only Cdc45 deletion
mutant that supported in vitro replication like wild-type was AIDR 169-208,
consistent with my in vivo complementation result. In contrast to the in vivo result,
76
Figure 12
A
B
66- 124-199-238336-
WT 69 126 201 240 337
+-+
-
+
-
ANI-ANI-ANI-
WT MR Pudrfed Cdc45
+-
+-
AC AC
AC
Pufied WT
Cdc45
DDK
-
DMK
ANI-697-448-311-
100 197 303 WT 438 660 680 660 Purified Cdc45
+ + +
DDK
Jim
00
antl4I
(Cdc45)
and-MCM
C
Purified
ANI-100
Purified
Cdc45 W
+
-
+
-DDK
Coomaessie stain:
DDK
ANI-100
94-
Purifed fom cuiyats
with Sk3 and SO
overexpression
(Cd-CS)
and-FLAG
(Cdc45)
anti-MCM-
antl-MCM-
anti-FLAG
Figure 12. Majority of Hsp70-bound Cdc45 mutants failed to support in vitro
replication. A. lpmol of FLAG-affinity purified Cdc45 wild-type and mutants were
tested for their ability to support in vitro replication, with -DDK as negative controls.
B. wild-type FLAG-purified Cdc45 is competent for replication but did not associate
with origin DNA in a DDK-dependent manner. Origin bound proteins were detected
by immunoblot using anti-Mcm2-7 (UM174) and anti-HA antibody (for Cdc45). C.
lpmol of Cdc45 wild-type and AN1-100 that were purified in presence of Sld3 and
Sld7 overexpression were tested for their ability to support in vitro replication as in
A. Origin bound proteins were detected by immunoblot using anti-Mcm2-7 (UM174)
and anti-FLAG antibody (for Cdc45).Coomassie stain of the purified Cdc45 AN1-100
was shown (Cdc45 band indicated by red arrow).
77
Cdc45 site specific mutants 66-69 and 124-126 also seemed to fully complement
Cdc45 function biochemically, even though Hsp70 was found to associate with 124126 (Figure 8B). On the other hand, mutant 336-337 only partially supported in
vitro replication (Figure 12A).
The interpretation of the in vitro replication result is complicated by two factors.
First, it is unclear how the associated Hsp70 chaperones affect Cdc45 structure and
function. Secondly, there is a challenge in distinguishing specific and non-specific
Cdc45 origin recruitment. I observed that both the wild-type and mutant Cdc45
purified proteins associated with origin DNA in a DDK-independent manner (Figure
12B & C). Neither titration of Cdc45 proteins, nor using high/low salt washes prior
to MNase/DNase treatment for DNA release from magnetic beads, effectively and
reproducibly removed DDK-independent Cdc45 DNA association. Remarkably,
addition of wild-type Cdc45 purified from lysates with overexpressed Sld3 and Sld7
reproducibly established DDK-dependent Cdc45 origin DNA recruitment in the in
vitro replication assay (Figure 12C). However, when similar strategy was applied in
the case of a Cdc45 truncation mutant (AN1-100), non-specific Cdc45 origin
recruitment was still observed (Figure 12C).
It has been known that ATP hydrolysis drives the cyclic process of Hsp70 substrate
binding and release. ATP-bound-Hsp70 binds substrate with low affinity, while
ADP-bound-Hsp70 or nucleotide-free-Hsp70 binds substrate with high affinity
(reviewed in Mayer & Bukau, 2005). To examine if addition of ATP prevented
78
Hsp70 association with Cdc45 mutants, I performed the purification of FLAG-tagged
Cdc45 mutant proteins in presence of excess ATP. After the cell lysates passed
through, I also thoroughly washed the column-bound protein with buffer containing
excess ATP. I observed that this additional ATP treatment was not effective in
removing Hsp70 contaminants in Cdc45 truncation mutants and a few site-specific
mutants such as 124-126. However, it was more effective for several site-specific
mutants. For example, 199-201, 336-337 and 485-488 mutants showed much
reduced Hsp70 contamination after the additional ATP treatment (Figure 13A & C).
Surprisingly, preliminary result showed that removal of Hsp70 from Cdc45 199-201
mutant by excess ATP washes supported the ability of Cdc45 199-201 mutant
complement in the in vitro replication assay (Figure 13B). However, removal of
Hsp70 from Cdc45 485-488 or 336-337 mutants did not allow these mutant
proteins to fully support in vitro replication (Figure 13D).
79
Figure 13
A
199-201
(Hsp7o)
B
1
2
DDK
+
-
Excess ATP
+
199-201
(ATP)
199-201
(Hsp70)
1
199-201
(ATP)
4
3
2
3
4
a-Cdc45
C
336-337
(Hsp70)
485-488
(Hsp70)
D
DDK
CExcess ATP
336-337
(ATP)
Excess ATP
4(5-488
(ATP)
-
+
336-33 7
(Hsp7O)
+
-
+
-
336-337 485-4
(ATP) (Hsp7O)
+.
-
485-488
(ATP)
Figure 13. Removal of Hsp70 in excess ATP purification condition affects in
vitro replication of Cdc45 199-201 mutant, but not 336-337 and 485-488
mutants. A & C. After excess ATP washes, most if not all Hsp70 were removed from
the Cdc45 mutant proteins. The eluted proteins from FLAG-affinity purification
(with or without excess ATP) were analyzed by 8% SDS-PAGE followed by
Coomassie staining. The red arrow points to the expected Cdc45 band, whereas the
lower bands were predicted to be Hsp70s. After excess ATP washes, a single Cdc45
band could be obtained. B. Addition of lpmol of Hsp70-free Cdc45 199-201 mutant
can fully complement Cdc45-dependent in vitro replication, whereas addition of
same amount of Hsp70-bound 199-201 mutant failed to complement in the same
assay. D. Addition of lpmol of either Hsp70-free or Hsp70-bound Cdc45 336-337
and 485-488 mutants failed to complement in the in vitro replication assay.
80
Discussion
How Cdc45 contributes to helicase activation remains unclear. In this study, I
investigated the function of ScCdc45 through mutational analysis. Using Cdc45 Nand C-terminal truncation mutants, I showed that both the N- and C-terminus of
Cdc45 are essential. Through targeted site-directed mutagenesis, I found that the
conserved motifs at the RecJ homology/DHH domain of Cdc45 are critical for Cdc45
function in vivo. Moreover, I identified a 61-amino-acid-long, charged IDR that is
dispensable but site-specific mutations within the region are lethal. Finally, I
mapped a region of ScCdc4S (amino acids 208-303) that appears to be important for
Cdc45 interaction with Sld7. Preliminary biochemical characterization of the Cdc4S
mutants was complicated by the observation that all the purified mutant proteins
were in a complex with Hsp70 chaperones. Future examination of biochemical
defects of the Cdc45 mutants isolated in this study could provide insights into the
role of Cdc45 in helicase activation.
Cdc45 may have an intrinsically disordered domain
Analysis of the Cdc45 amino-acid sequence using the PONDR VL-XT program
identified a region of Cdc45 that is strongly predicted to be intrinsically disordered
(Figure 1). Disordered regions can undergo disorder-to-order transitions upon
binding to one or more partners. This type of transition is commonly used for
signaling and protein-protein interactions (reviewed in Orosz & Ovidi, 2011). Given
that Cdc45 has multiple interacting partners, it is possible that this domain could
play a role in one of more of these interactions. My finding that a Cdc45 mutant
81
lacking the putative IDR is viable in vivo argues against such a molecular recognition
hypothesis. It remains to be verified whether the putative IDR is disordered
experimentally. For example, one could analyze the thermal melting of Cdc45
domains by Fast parallel proteolysis (FASTpp). FASTpp reveals transitions between
folded and unfolded protein states, by parallel exposure of protein samples to a
wide range of different temperatures, in the presence of a thermostable protease
such as thermolysin (Minde et al. 2012).
Hsp70 interaction with mutant Cdc45 proteins
Affinity purification of one of the mutant Cdc45 protein (AC448-650) followed by
MS analysis revealed abundant association of Ssal/2 family yeast Hsp70 (70 kDa
heat shock protein) chaperones, as well as co-chaperones such as Ydj1 and Sisi
(Figure 8 &Table 2). Surprisingly, these Hsp70s were observed to co-purify with all
Cdc45 inactivating mutants, suggesting the mutations result in significant alteration
of Cdc45 structure or partial unfolding of the proteins. Hsp70 association was not
observed with wild-type Cdc45 or viable Cdc45 mutants such as the IDR deletion
mutant (AIDR 169-208) (Figure 8B). These observations suggest a correlation
between Cdc45 function and Hsp70 binding. It is possible that a slight perturbation
of Cdc45 folding can result in significant change in its structural conformation,
leading Hsp70 binding and concomitant loss of Cdc45 activity. It remains unclear
whether similar interactions are occurring when the mutant proteins are present at
low levels.
82
The observation that mutant phenotypes correlate with Hsp70 association is not
limited to Cdc45. It has been reported that a subset of p53 mutants but not wild-
type, specifically bind to Hsp70 with varying degree of affinity (Wiech et al. 2012;
Gaiddon et al. 2001; Gannon et al. 1990; Pinhasi-Kimhi et al. 1986). Cell culture
study of mutant p53 suggests that Hsp70 enhances formation of cytoplasmic
aggregates and stabilizes the half-life of mutant p53 proteins (Wiech et al. 2012).
Applying a similar analogy to Cdc45 mutants, it is possible that the Hsp70
association itself accounts for the failure of Cdc45 mutants to complement the loss
of Cdc45 in vivo. For instance, Hsp70 binding potentially interferes with Cdc45
nuclear localization. To verify whether solubility of mutant Cdc45 is affected by
Hsp70, confocal microscopy analysis coupled with immuno fluorescence or direct
visualization of GFP-tagged Cdc45 mutants could be performed. If Hsp70 association
leads to Cdc45 aggregation and alteration of its subcellular distribution, I would
expect mutant Cdc45 to be sequestered in cytoplasm forming visible spots instead
of having diffuse nuclear localization as previously described in wild-type Cdc45
(Hopwood & Dalton, 1996).
Known mechanisms by which Hsp70-chaperones assist protein folding might shed
light on how Hsp70 association affects Cdc45 mutants. Hsp70 can recognize and
specifically bind to hydrophobic segments of mature, partially unfolded proteins in
an ATP-hydrolysis-dependent manner. Hsp70 in an ATP-bound conformation binds
to its substrates with low affinity. Association of co-chaperones with Hsp70 can
direct Hsp70 to specific substrates and stimulate ATP hydrolysis of Hsp70, thus
83
inducing Hsp70 substrate binding (reviewed in Mayer & Bukau, 2005).
Mechanistically, there are at least two modes for Hsp70 action to facilitate
disaggregation and proper folding of its bound substrates (reviewed in Mayer &
Bukau, 2005): (1) passive mode or "kinetic partitioning": Hsp70 transiently binds to
partially unfolded regions of a substrate and keep the level of free substrate low to
prevent aggregation. The free substrates are able to fold by themselves or maintain
its biologically relevant conformation structure; (2) active mode or "local
unfolding": Hsp70 repeatedly binds to protein substrates that have already formed
aggregates to induce local unfolding or conformational remodeling. In this active
mode, ATP hydrolysis is required for the repeated binding process and energy of
ATP is needed to mediate refolding of substrate to native state.
Models to explain the effect of Hsp70 action on Cdc45 could use either the active
and passive modes. First, Hsp70 binding did not eliminate mutant Cdc45 molecular
function entirely. This was indicated by the ability to detect Sld3 and Sld7
interaction with Hsp70 associated Cdc45 deletion mutants (Figure 10), as well as
the ability for some Hsp70-binding mutants (such as 336-337, 124-126) to at least
partially support in vitro replication (Figure 12 & 13). To account for such
observation, it is possible Hsp70 works in a passive mode to prevent Cdc45
aggregation and Cdc45 structural conformation is largely maintained. Alternatively,
Hsp70 could work in an active mode to facilitate disaggregation and mediate
refolding, so that a subpopulation of the Cdc45 mutants could refold to their
biologically relevant structural conformations.
84
Secondly, different modes of Hsp70 action could also explain why removal of Hsp70
from Cdc45 mutants by ATP treatment might or might not affect their ability to
complement biochemically. In the case of Cdc45 199-201 mutant, removal of Hsp70
efficiently restored in vitro replication (Figure 13B). This implies that 199-201
probably had a functional conformational structure but the passive binding of
Hsp70 at the unfolded region is inhibitory to its normal function. In contrast,
removal of Hsp70 from Cdc45 485-488 and 336-337 mutants did not facilitate in
vitro replication (Figure 13D). There is no clear explanation for this result. It is
possible that preventing Hsp70 binding in an active mode leads to irreversible
aggregation of Cdc45 mutants, or the exposed, unfolded region(s) caused loss of
essential interactions. To determine if it was aggregation of Cdc45 mutants that
accounts for failure to complement in vitro replication, I could perform gel filtration
on these complexes to see if they migrated in the void of the column. If I separated
out monomeric forms in this process, I could test the soluble mutant proteins for
their ability to restore in vitro replication.
Although the co-purification of co-chaperones suggest that Hsp70 likely associates
with the isolated Cdc45 mutants to facilitate folding, I cannot exclude the possibility
that Hsp70 chaperones might play other functional roles. For example, it has been
reported that bacterial Hsp70 contributes to DnaB helicase activation in lambda
DNA replication (Zylicz, 1993). It would be interesting to examine any association of
Hsp70 with wild-type Cdc45 by MS analysis.
85
Role of the N-terminus of ScCdc45
The preliminary expression data shows that site-specific mutations at N-terminus of
Cdc45 (including the RecJ homology region) leads to no or unstable Cdc45 protein
expression. Low protein expression of mutants, but not loss of function, might
account for the failure to support growth in these N-terminal mutants. One
possibility is that these N-terminal site-specific mutations render increased
susceptibility to protease cleavage leading to protein degradation. It will be
important in the future to determine the level of expression of all of the mutant
proteins when expressed from the endogenous Cdc45 promoter. A piece of evidence
in favor of this model is that purified Hsp70-free 66-69 and Hsp70-bound 124-126
complement Cdc45 function in the in vitro replication assay. Although these mutants
are proposed to have insufficient Cdc45 expression to support DNA replication in
vivo, in vitro this issue could be circumvented. This could be because of the lack of
proteolysis due to addition of protease inhibitors, or because the amount of Cdc45
added in the replication assay is well above the amount required. Another idea to
resolve the discrepancy between my in vitro and in vivo results could be the
overexpression of limiting factors such as Sld3, Sld2 and Dpb11 in the in vitro
replication assay. Since these proteins are known to interact with Cdc45, their highcopy number might result in suppression of certain Cdc45 mutations.
If the low expression of the N-terminal site-specific Cdc45 mutants is mainly due to
protein stability problems, there are potential ways to improve stability of these
mutant proteins for better protein expression. Instead of purifying mutants in yeast,
86
mutants could be purified in other cells such as an E coli. A cell-free in vitro
translation system might overcome sensitivity to proteases. Alternatively, since
epitope tags used in my study are on the C-terminus, I could consider fusing a
relatively large protein tag such as GST at the N-terminus to see if protein stability is
improved. Intriguingly, SDS-PAGE analysis of purified Cdc4S wild type protein
occasionally reveals a Cdc45 doublet that is still visible after immunoblotting
against C-terminal tag (Figure 12B). This suggests there is a population of Cdc45
that is either post-translationally modified or N-terminally truncated.
Coincidentally, purification of human wild-type cdc45 with FLAG tagged at Nterminus also reported a Cdc45 doublet (Kang et aL 2012). In that study, the doublet
was proposed to be due to proteolysis.
Based on preliminary expression data of Cdc45 mutants, I hypothesize that the
Cdc45 N-terminus is important for its protein stability. Mutations at the Cdc45 N-
terminus might partially unfold Cdc45, leading to an increased susceptibility for
proteolysis. To address if the low expression of N-terminal Cdc45 mutants confers
lethality, future experiments could determine if these proteins also show low or no
expression under endogenous Cdc45 promoter in vivo. It would also be worth
determining if these mutations can complement when they are overexpressed.
It is also possible that mutations within the Cdc45 N-terminus result in failure to
interact with another protein partner and that this interaction is important for
Cdc45 stability. Interestingly, purified GST-tagged human Cdc45-1-303 shows direct
87
interactions with TopBP1 (in yeast, Dpb11; Schmidt et al. 2008). A previously
published ScCdc45 dominant mutation JET-1 is also located at the N-terminus
(H22Y) and increases the affinity of Cdc45 binding to Sld3 and Dpb11 (Tanaka S,
2007).
Evolutionary link between Cdc45 and RecJ
Cdc45 has a sequence similarity to bacterial and archaeal DHH family
phosphoesterases, but the importance of this evolutionary link in Cdc45 function is
not clear. In my study, the RecJ homology region identified based on HHpred
analysis is limited to N-terminus (a.a.8-128) of ScCdc45 (Figure 3). On the other
hand, by combining various sequence alignment and threading algorithms a recent
study reported that there is similarity between RecJ and the entire human Cdc45
(Krastanova et al. 2011). In that study, two unique eukaryotic sequence insertions in
Cdc45 that are absent in bacterial Recj were identified. Remarkably, considering the
multiple sequence alignment result published in the study (Krastanova et al. 2011),
three of my site-specific mutants lie within their identified Reci motifs (35-37 in
motif I; 124-126 in motif III and 238-240 in motif Ila). Moreover, the IDR (including
mutations 171-173 and 199-201) is within a unique eukaryotic insertion that is
absent in archaea and bacterial RecJ. The 336-337 mutant lies within another
unique insertion that is absent in bacterial RecJ but present in archaea. Taken
together, Recj motifs might be important for Cdc45 function and the two insertion
regions probably evolved to play a novel role in Cdc45.
88
Role of the C-terminus of ScCdc45
The only C-terminal mutant that was isolated in my study is a ts mutation 485-488.
Preliminary result shows both Hsp70-bound or ATP-treated 485-488 mutant failed
to complement replication in vitro (Figure 13D), suggesting the mutation lacks at
least one essential DNA replication function. Interestingly, a recent paper showed
that the C-terminus of Cdc45 is required for ssDNA binding and mutating multiple
positively charged sites at C-terminal region results in HU sensitivity (Bruck &
Kaplan, 2013). In my study, 485-488 mutant, however, did not confer HU sensitivity
(Figure 6B). It would be interesting to test whether 485-488 mutant has a defect in
ssDNA binding using EMSA. In a genetics study, ScCdc45 mutation (cdc45-1 0)
locating at the C-terminus (G510D) was shown to be defective for S phase
progression and hypersensitive to camptothecin (Cpt) that targets DNA
topoisomerase I (Reid et al. 1999). It remains to be determined whether the Cterminus of Cdc45 plays a role in replication stress and DNA damage response.
Cdc45 interaction with Sld3 and Sld7
Consistent with published results, I was able to detect co-immunoprecipitation of
Sld3, Sld7 with Cdc45 but important controls need to be done to verify this result.
The estimated stoichiometric ratio of Sld7: Sld3 in a complex with Cdc45 is
approximately 2:1 (Figure 9A). However, the amount of Sld3 and Sld7 associated
with wild-type Cdc45 was far below the detection limit of Coomassie staining even
though Cdc45 was easily detected (Figure 9B). It is unclear why a stoichiometric
Sld7-Sld3-Cdc45 complex was not readily isolated from soluble extract. One
89
possibility is that the complex is not stable in solution (e.g. off the DNA). Consistent
with the interaction being unstable, Sld3 and Cdc4S have been shown to form a
complex throughout the cell cycle, but only in presence of an in vivo crosslinking
agent (Kamimura et al. 2001). Also, it is possible that post-translational modification
contribute to stabilization of Sld7, Sld3 and Cdc45 complex and the enzymes
responsible are not abundant enough to modify the overexpressed proteins.
Alternatively, the interaction may require Mcm2-7. Biochemical studies showed that
individually purified Sld3, Cdc45 and Mcm2-7 form a stable, ternary CMS complex
(Bruck & Kaplan, 2011). It is also possible that there is inhibitory activity in the
soluble fraction (for instance, the overexpression of Sld7 itself or Rad53
phosphorylation of Sld3) that inhibits the Sld3-Cdc45 interaction. It would be
interesting to individually purify Sld3, Sld7 and Cdc45, then incubate Cdc45 with
Sld3 in presence or absence of Sld7 followed by IP analysis and gel filtration. This
would remove possible inhibitory activity from cell lysate and address how Sld7
affect Cdc45 and Sld3 interaction.
It is known that Sld3-Sld7 and Cdc45 chromatin recruitment is dependent on DDK
activity. How DDK phosphorylation of Mcm2-7, which presumably changes MCM
conformation, facilitates recruitments of Cdc45 and Sld3-Sld7 is not clear. It is
possible that one or more proteins in a Sld7-Sld3-Cdc45 complex binds directly to
phosphorylated sites on Mcm2-7. Alternatively, DDK phosphorylation could alter
the conformation of the loaded Mcm2-7 (e.g. dissociating the double hexamer into
two single hexamers) and this change provides a new surface for Sld3-Sld7-Cdc45
90
binding. Although it has been shown that recruitment of Sld3 and Cdc4S is codependent (Heller et al. 2011, Kamimura et al. 2001), it is still possible that Cdc45
and Sld3/Sld7 are recruited separately since the interaction between Sld3 and
Cdc45 is not robust (Tanaka S. 2011) but only stabilized when both bound to
chromatin.
In my study, I observed Sld7 interaction with Cdc45 deletion mutants A1-197 and
A169-208 but not in A1-303 (Figure 10A & C), suggesting that amino acids 208-303
of Cdc45 are important for Sld7 interaction. Interestingly, the disappearance of Sld7
band in A1-303 is accompanied with change in banding pattern of putative Sld3
(Figure 10C), possibly due to increased proteolysis. This implies that the same
region might also be important for Sld3 interaction.
In budding yeast, Sld7 is not essential but is still important for cell growth (Tanaka
T. et al 2011). Sld7 has been shown by yeast two-hybrid to interact with N-terminal
end of Sld3. In Asld7 cells, Sld3 protein levels are reduced and the cells are sensitive
to HU (Tanaka T et al, 2011). Based on these findings, the authors speculated that
Sld7 alters conformation of Sld3 to increase its resistance to proteases. My
preliminary experimental observation are consistent with their hypothesis.
Intriguingly, Cdc4S binds to Sld3 alone more efficiently than Sld3-Sld7, suggesting
that Sld7 reduces Sld3 association with Cdc45 (Tanaka T. et al 2011). This
observation led to the proposal that Sld3 alter its affinity for Cdc45 by interacting
91
with other proteins. Consistent with this hypothesis, in absence of Sld7 association
in Cdc45 A1-303 there seem to be a higher amount of Sld3 co-purified with Cdc45
that was visible by Coomassie staining (Figure 10B). One hypothesis could be that
Sld7 interacts to facilitate dissociation of Sld3 from Cdc45. This might be important
for subsequent binding of GINS, since protein-protein interaction study implicated
that GINS and Sld3 compete for Mcm2-7 binding (Bruck & Kaplan, 2011).
Interestingly, a recent study reported a potential human homolog for Sld7-MTBP
and showed that MTBP is important for CMG assembly using DNA fiber labeling and
RNAi depletion (Boos et al. 2013).
Potential function of the Cdc45 IDR
Although the Cdc45 IDR is not essential in vivo, two mutations of the negatively
charged patches are lethal. Moreover, in the IP study of Cdc45 AIDR, Sld3 may be
phosphorylated. However, further experiments such as phosphatase treatment will
be required to verify that the change in Sld3 mobility is due to phosphorylation.
Taken together, these observations suggest that deletion of the Cdc4s IDR bypasses
an inhibitory function that is related to Sld3 phosphorylation. Consistent with the
observed Hsp70 association with Cdc45 199-201 (Figure 13A), site-specific
mutations within the IDR might lead to a change of Cdc45 folding that interferes
with Cdc45 function. To test if the charge of IDR plays an important function, it
would be interesting to substitute the negative charges with positive ones and see if
a more severe defect is observed.
92
Future characterization of Cdc45 mutants
For future experimental studies, I would examine whether the ScCdc45 mutants in
my study form aggregates both in vivo and in vitro. This would help to determine
whether the ScCdc45 mutants maintain physiologically relevant structural
conformation. Although results of my co-IP study and in vitro replication assay
revealed that at least some of the Cdc45 functions were retained in my Cdc45
mutants, the result would be more convincing if the purified Cdc45 mutant proteins
are disaggregated and more or less homogeneous. Controlled amount of soluble
Cdc45 mutant proteins would then be tested in my in vitro replication assay using
new S phase extracts that have key replication initiation proteins (such as Sld3, Sld7,
Sld2, Dpbll, Mcm1O, Dpb2, Psf2 etc) epitope tagged. If the issue of non-specific DNA
binding of Cdc45 persists in the in vitro replication assay, Sld3 origin recruitment
could serve as an additional control, as the recruitment of Sld3 and Cdc45 has been
shown to be co-dependent (Heller et al. 2011; Kamimura et al, 2001). It would also
be interesting to test if Cdc45 origin recruitment could be reconstituted by adding
purified Cdc45, Sld3 and Sld7 to loaded Mcm2-7 in presence of DDK activity in an in
vitro assay. If this is possible, the interactions among these proteins could be
examined in greater detail using single-molecule FRET analysis.
93
Materials and Methods
Strain construction
The yeast strains used in this study are listed in Supplemental Table 1. Plasmid
constructs were generated by standard laboratory methods, followed by yeast
transformation.
In vivo complementation analysis
A Cdc45 swapper strain was generated first by replacing endogenous CDC45 with
KanMX in a diploid strain. A non-integrative plasmid with a URA3 marker and wildtype CDC45 under endogenous CDC45 promoter and 3'UTR was transformed into
the strain. After sporulation and tetrad analysis, haploid cdc45A-KanMX spores
carrying Ura-marked wild-type CDC45 plasmid were identified. LEU-marked cdc45
mutants with and without C-terminal epitope tag under control of endogenous
CDC45 promoter and 3'UTR were integrated into the swapper strain. The URA3marked wild-type CDC45 plasmid was selected against by plating on 5-FOA. Mutants
were then screened for complementation by their ability to grow in presence of 5FOA.
Protein purification
Both wild type and mutant Cdc45 tagged with 3XHA and 3X FLAG were expressed in
yeast cells (yRH101, pep4A barlA background) and purified with anti-FLAG M2
agarose beads (Sigma) as described in Sigma protocol. Asynchronous cell extracts
were incubated with FLAG resin for 4 hours at 4C either in presence or absence of
94
5mM ATP. The bound proteins were washed in presence or absence of 2mM ATP
(300mMKGlu/HEPES Washing buffer: 300mM Potassium glutamate/KGlu; 25mM
Hepes, pH = 7.6). Finally, 300mMKGlu/HEPES elution buffer containing 0.15mg/ml
of triple FLAG peptides were used to for release of bound FLAG-tagged proteins.
Cdc45-dependent in vitro replication assay
Replication assays were performed on 7.6 kb linear DNA template containing wildtype ARS1 replication origin. The linear DNA template was prepared by cutting
pARS1/Nco-Nco with Sad and BamHI, followed by a fill-in reaction with dCTP,
dTTP, dGTP and Biotin-14-dATP. The biotinylated linear fragments purified with
Qiagen PCR purification kit and coupled to streptavidin-coated magnetic beads.
Beads coupled to 125 fmol of DNA were used in each replication assay. Helicase
loading and DDK reaction were performed as previously described (Heller et al.
2011), only the final replication reaction involving S-phase extract was modified.
The S-phase extract used in these studies only overexpressed Sld2, Sld3 and Dpb11
(not Cdc45). Addition of 1 pmol of purified, wild-type Cdc45 along with S phase
extract in the previously described replication reaction (Heller et al. 2011) supports
in vitro replication. Replication products were analyzed by alkaline gel
electrophoresis. The resulting gel was dried and analyzed by exposure to a
phosphoimager screen. Proteins bound to the DNA template were analyzed by SDSPAGE followed by immunoblotting to the indicated proteins (Heller et al. 2011).
95
Supplemental Material
Supplemental Table 1. Yeast strains used in this study. All strains are in the
W303La background (MA Ta ade2-1 ura3-1 his3-11,15 trpl-1 leu2-3,112, cani-100,
lys2::HisG).
Strain
Name
yRH101
yBC018
Major Gene
Involved
BAR1, PEP4
Wild-type CDC45
yBC002
CDC45(A Ni-100)
yBC003
CDC45(A Ni-197)
yBC004
CDC45(A Nl-303)
yBCOO5
CDC45(AN -438)
yBC006
CDC45(AN -590)
yBC007
CDC45(AC597-650)
yBC008
CDC45(AC448-650)
yBC009
CDC45(AC311-650)
yBC010
CDC45(AC204-650)
yBC011
CDC45(ACl09-650)
yBC019
CDC45 35-37
yBC020
CDC45 66-69
yBC021
CDC45 124-126
yBC025
CDC45 171-173
yBC027
CDC45 199-201
yBC028
CDC45 238-240
yBC031
CDC45 336-337
Genotype
barl::HisG,pep4::kanMX
bar::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(full-length)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(A1-100)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(A1-197)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(Al-303)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(A1-438)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(Al -590)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(A597-650)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(A448-650)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(A311-650)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(A204-650)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(Al 09-650CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(35-37)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(66-69)CDC45-3HA3FLAG
bar::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(124-126CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(171-173)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(1 99-201)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(238-240)CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10-
96
(336-337)CDC45-3HA3FL AG
yBC034
CDC45 485-488
yBC036
CDC45(A169-229)
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(485-488CDC45-3HA3FLAG
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10(Al 69-229)CDC45-3HA3FLAG
yBC017
yBC038
yBC070
I
CDC7, DPB11, SLD2,
SLD3, CLB5, CDC28,
barl::HisG, pep4::Hph, NA T::MCM10-13myc
his3::Cdc45-3HA,cdc7-4, leu2::GAL1,10-DPBl1
MCM10
lys2::GAL1,10-Sld2/SId3, ura3:: GAL1,10-Cdc28/Clb5
CDC45
CDC45, SLD3, SLD7
cdc45::kanMX,pRS416-pCDC45-CDC45
barl::HisG,pep4::kanMX, leu2::pAS584-GAL1,10CDC45-3HA3FLAG, his3::pRS403-GAL1,10-Sld3I_V5/Sld7-V5
97
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