Loading of cohesin and cohesion generation in
response to DNA damage.
Rutger.C.C. Hengeveld
The sister chromatids are held together by the conserved cohesin complex from DNA
replication in S phase until the metaphase to anaphase transition during mitosis.
Generation of sister chromatid cohesion is essential for creating bipolar attachments
between chromosomes and microtubules to allow equal segregation of sister
chromatids in anaphase. In addition, the cohesin complex plays important roles in DNA
damage checkpoint signaling and repair of DNA double strand breaks. In mammalian
and yeast studies it was shown that the cohesin complex and its important loading
factors are recruited to sites of DNA damage. Recent yeast studies indicate that not
only local cohesion is generated at the damaged site. Remarkably, DNA damage
triggers genome-wide cohesion establishment. However, it is still unknown if this is
similar in mammalian cells. Phosphorylation of the cohesin complex components,
SMC1 and SMC3, plays important roles in DNA damage induced checkpoint signalling
in human cells. Several post-translational modifications mediate the different functions
of the cohesin complex. Here, the role of the cohesin complex during DNA damage and
its post-translational modifications are discussed, with a specific focus on the
differences between yeast and human.
The cohesin complex
Scc3 is associated with the complex via
From DNA replication in S phase until the
Scc1 (Nasmyth and Haering 2005). The
metaphase
in
cohesin complex is believed to hold the
mitosis, chromatid pairs are held together by
sisters chromatids together by co-entrapping
the cohesin complex. The establishment of
them inside its ring-shaped structure which
cohesion in S phase and the removal in
allows entrapment of the sister chromatids.
to
anaphase
transition
mitosis is mediated by accessory proteins
and post-translational modifications of the
Loading of the cohesin complex
cohesin subunits. The core of the cohesin
The conserved proteins Scc2 and Scc4 are
and the similar condensin complexes are
responsible for loading of cohesin during
build
of
telophase in higher eukaryotes (Ciosk et al.
are
2000, Tomonaga et al. 2000 and Bernard et
conserved from yeast to human (Hirano et
al. 2006). The mechanism by which Scc2
al. 2006). In eukaryotes, the SMC protein
and Scc4 recruit cohesin to the DNA is
family consists of six members, SMC1-6,
largely unknown. However, the recruitment
and are between 1000-1300 amino acids in
of Scc2 and Scc4 to DNA appears to be
length. All SMC proteins contain two large
dependent on the pre-replication complex
coiled coil domains between a central hinge
(pre-Rc) in Xenopus egg extracts (Gillespie
and a globular domain (Hirano et al. 2006)
and Hirano et al. 2004 and Takahashi et al.
(Fig. 1A). Because the protein folds back at
2004). The pre-RC forms by assembly of the
the central hinge domain, the N and C
origin of replication recognition complex
terminal heads are in close proximity
(ORC), Cdt1 and Cdc6 during the initiation
creating
domain
step of DNA replication (Bell and Dutta et al.
(Nasmyth and Haering 2005). The hinge
2002) (Fig. 1B). This complex in turn recruits
domain also facilitates dimerization of SMC
the Mcm2-7 helicase to the DNA in an
molecules creating a V-shaped structure.
inactive form. The activation of the Mcm2-7
Electron microscopy studies showed that
helicase requires phosphorylation by the
the SMC molecules are approximately 50
kinase Cdc7 and interaction with Cdc45 and
nm in length and the hinge domain is
Gins (Ilves et al. 2010). Importantly, Cdc7
extremely flexible (Haering et al. 2002 and
kinase interacts with Scc2/Scc4 and its
Gruber et al. 2003). Three different SMC
kinase activity is required for efficient
hetero-dimers
recruitment of the complex to the DNA
from
Structural
Chromosome
(SMC)
a
(cohesin),
functional
are
SMC2/4
Maintenance
proteins
ATPase
formed:
and
SMC1/3
(condensin)
and
(Takahashi et al. 2008) (Fig. 1B). However
SMC5/6. The cohesin complex consists of
the
SMC1, SMC3 and the non SMC proteins
association of cohesin with chromatin in
Scc1,
Pds5, Sororin and Wapl
yeast, indicating that in yeast cohesin is
(Losada et al. 2000, Sumara et al. 2000,
recruited to chromatin in a DNA-replication
Haering et al. 2002 and Gruber et al. 2003).
independent manner. Whether the pre-Rc is
The N- and C-terminal domains of SMC1
required for cohesin loading in mammalian
and SMC3 are coupled via Scc1 (Fig. 1A).
cells is unclear.
Scc3,
pre-Rc
is
not
essential
for
the
Figure 1| Cohesin complex and cohesin loading. (a) The cohesin complex consisting of SMC1 (green), SMC3
(red), Scc1 (blue) and Scc3 (gray). The coiled coils and ATPase domains of SMC1 and SMC3 are highlighted. (b) In
Xenopus egg extracts, the loading of Scc2 and Scc4 is dependent on the pre-replication complex and Cdc7 kinase
activity.
Establishment of cohesion in S phase
to Pds5 through FGF sequence motifs and
The recruitment of cohesin to DNA is
interacts with Scc1 and Scc3. How SMC3
essential but not sufficient to entrap the
acetylation exactly counteracts Scc3, Pds5
sister
generation
and Wapl activity is still unknown. One
requires passage trough S phase and the
model is that, at least in animal cells,
action of establishment factors (Uhlmann
acetylation of SMC3 recruits a cohesion
and Nasmyth 1998). The expression of a
maintenance factor, called sororin, which
non-cleavable Scc1 after DNA replication
also contains FGF sequence motifs. When
does not hinder anaphase, indicating that
sororin is recruited it displaces Wapl from its
cohesion is not generated after S-phase
binding
(Haering et al. 2004). The conserved
cohesion
acetyltransferase, Eco1/Ctf7, is essential to
(Nishiyama et al. 2010) (Fig. 2). However,
establish sister chromatid cohesion. The
thus
human
Eco1
Importantly, in human cells, depletion of
both
Wapl or Pds5 can rescue the phenotype of
chromatids.
genome
orthologs,
Cohesion
encodes
Esco1/Esco2,
two
and
are
to
far
Pds5
and
generation
no
maintenance
sororin
is
(Hou and Zou 2005). In yeast studies, two
expression of a non-acetylatable SMC3
conserved lysines (K112 and K113) within
mutant,
the ATPase domain of SMC3 were identified
mechanism is conserved from yeast to
as major Eco1 targets in S phase (Rolef
human (Gandi et al. 2006 and Terret et al.
Ben-Shahar et al. 2008, Zhang et al. 2008,
2009).
cohesion by counteracting a so far poorly
understood
‘’anti-establishment
activity’’
associated with Scc3, Pds5 and Wapl
(Tanaka et al. 2001, Rowland et al. 2009
and Sutani et al. 2009) (Fig. 2). Wapl binds
indication
deficiency
known.
Esco1
This acetylation allows establishment of
Esco2
of
allows
involved in the establishment of cohesion
Unal et al. 2008 and Rowland et al. 2009).
and
yeast
thereby
that
the
or
the
regulatory
Figure 2| Generation of cohesion during S phase. At S phase onset the cohesin complex is tightly bound to Pds5
and Wapl which antagonize cohesion establishment. When SMC3 is acetylated by Eco1, sororin is recruited and
displaces Wapl from its binding to Pds5 and thereby allows cohesion generation or maintenance.
Cohesin removal during mitosis
pathway in human cells remains to be
Cohesin is removed during mitosis by two
established. One explanation could be that
steps. The first, also called the prophase-
the human chromosomes are larger than
pathway, removes the bulk of cohesin from
yeast chromosomes. During the metaphase
chromosome arms. This removal depends
to anaphase transition there is a lot of
on the action of Wapl and Polo-like kinase-1
pulling force from the spindle poles to the
(Plk-1), which is believed to phosphorylate
chromosomes which potentially could break
the cohesin subunit, Scc3 (Hauf et al. 2005,
the chromosomal arms when they are still in
Waizenegger et al. 2000 and Kueng et al.
the cohesive state.
2006). How Wapl facilitates the removal of
chromosomal arm
cohesin is currently
DNA damage response and checkpoint
unknown. Cohesin is protected at the
recovery
centromeric regions by the activity of Bub1
The DNA is constantly exposed to external
kinase which phosphorylates H2A and
and internal factors which cause DNA
thereby recruits proteins of the Shugoshin
damage. The most common insults are
family (Sgo1 and Sgo2) to the inner
ionizing radiation, chemicals, and oxidative
centromeric region (Kawashima et al. 2010).
stress which create DNA double strand
Sgo1 is thought to protect centromeric
breaks. A functional DNA damage response
cohesin by recruiting Protein Phosphatase
stops the cell cycle progression until the
2A (PP2A) which may dephosphorylate
damage is repaired.
Scc3 (Hauf et al. 2005, Riedel et al. 2006
process to maintain genome stability and
and Kitajima et al. 2006). The remaining
thus
Cohesin that persists at centromeric regions
cancerous (Hoeijmakers et al. 2001 and
is removed at the metaphase to anaphase
Lobrich et al. 2007). When cells undergo
transition by cleavage of Scc1 by the
DNA damage, specialized ‘sensor’ proteins
protease separase (Peters et al. 2006).
detect the site of DNA damage and activate
Importantly, in yeast, all cohesin remains on
the DNA damage response pathway which
the chromosomal arms until cleavage by
results in a cell cycle arrest (Fernandez-
separase. The function of the prophase-
Capetillo et al. 2003). Then the damage can
to
protect
This is an essential
cells
from
becoming
be repaired by non-homologous end-joining
autophosphorylation (ATM is converted from
(NHEJ) or by homologous recombination
an
(HR) (Ohnishi et al. 2009 and San Filippo et
monomers),
al. 2008). During NHEJ, the broken ends are
complex acts upstream of ATM (Fig. 3A)
directly re-ligated and often the double stand
(Falck et al. 2005). The C-terminus of Nbs1
breaks are repaired accurately. However,
interacts with the so called HEAT repeats of
imprecise repair also frequently occur during
ATM to mediate this recruitment (You et al.
NHEJ due to the loss of nucleotides and
2005). Activated ATM first phosphorylates
thus loss of genetic information. Therefore
H2AX on a conserved serine residue (serine
HR is performed during S and G2 phase,
139)
using a homologous template from the sister
phosphorylated: H2AX is called γH2AX),
chromatid (San Filippo et al. 2008) Since the
creating a docking site for the mediator of
sister chromatids are identical this will
DNA damage checkpoint 1 protein (MDC1)
accurately
genetic
(Fig. 3A) (Stucki et al. 2005; 2006). Kinetic
material. When cells are not able to repair
studies showed indeed that first the MRN
the damage properly the cells will die by the
complex and MDC1 are accumulated at site
induction of apoptosis.
of DNA damage (Lukas et al. 2004). Other
preserve
the
loss
inactive
at
dimer
into
indicating
the
two
that
the
C-terminus
active
MNR
(when
important factors like breast cancer type-1
Formation of a wide γH2AX region
susceptibility protein (BRCA1) and 53BP1
The central kinases involved in the DNA
(discussed
damage response are the phosphoinositide
suggesting that the recruitment of the MRN
3-kinase related kinases (PIKKs); ATM and
complex and MDC1 is an early event in the
ATR (Bartek and Lukas 2007 and Jackson
DNA damage response. In addition, since
and Bartek 2009). The substrates of these
the recruitment of Nbs1, BRCA1 and 53BP1
kinases mediate cell cycle arrests in G1, S
failed in cells lacking MDC1 it is proposed
and G2, facilitate DNA repair and can
that
eventually induce apoptosis (Shiloh et al.
platform to accumulate proteins involved in
2003, Bakkenist et al. 2004, Bartek et al.
the DNA damage response (Lukas et al.
2004 and Lukas et al. 2004). The MRN
2004 and Bekker-Jensen et al. 2005).
complex consisting of Mre11, Rad50 and
Importantly,
Nbs1 is the major sensor of DNA damage.
region containing multiple conserved acidic
This multifunctional complex is involved in
motifs (SDT motifs) are phosphorylated by
DNA
telomere
casein kinase 2 (CK2) on both serine and
checkpoint
threonine residues (Spycher et al. 2008,
signaling (Petrini et al. 2003). In response to
Melander et al. 2008 and Chapman et al.
DNA damage the MRN complex recognizes
2008). This phosphorylation mediates the
the free DNA ends of the DNA double strand
protein-protein interaction between MDC1
breaks
When
and the N-terminal FHA domain of NBS1.
positioned, the MRN complex recruits ATM
This interaction is essential for accumulation
which
and retention of the MRN complex and ATM
repair
maintenance
mechanisms,
and
(Stucki
then
cell-cycle
et
is
al.
2006).
activated
by
below)
MDC1/γH2AX
after
are
recruited
interaction
MDC1
later,
forms
recruitment
a
a
at double strand breaks (Fig. 3A). This
53BP1 remains unknown. However, recently
newly
it
recruited
ATM
phosphorylates
was
shown
that
the
histone
proximal H2AX molecules. Repetition of
methyltransferase
MMSET
these events creates a positive feedback
phosphorylated
loop that leads to the formation of a wide
phosphorylated form can directly bind to the
γH2AX region around double strand breaks
BRCT domain of MDC1. After its recruitment
(Fig. 3A) (Stucki et al. 2006).
MMSET methylates H4 on lysine 20, which
by
ATM
is
and
its
facilitates 53BP1 recruitment (Pei et al.
Recruitment
53BP1
and
the
BRCA1
2011) (Fig. 3B).
complex to sites of DNA damage
The N-terminus of MDC1 is phosphorylated
Checkpoint response
by ATM on the so called TQXF motifs and
The increased local concentration of ATM,
serves as recruitment factor for RNF8
BRCA and 53BP1 allows phosphorylation of
(Mailand et al. 2007) (Fig. 3A). RNF8
Chk2 on serine 68 by ATM (Matsuoka et al.
contains a RING finger domain at the C-
2000). Chk2 is a protein kinase that triggers
terminus which provides its E3 ubiquitin
a cell cycle arrest after its activation (Fig.
ligase activity and a FHA domain at the N-
3C). One of its major targets is p53 which is
terminus
the
highly unstable in undamaged cells. p53
phosphorylated form of MDC1 (Fig. 3B)
normally binds to the ubiquitin ligase MDM2,
(Mailand et al. 2007, Kolas et al. 2007 and
which targets p53 for destruction by the
Huen et al. 2007). The ubiquitin cofactor
proteasome (Momand 1992 et al., Oliner
UBC13 binds to RNF8 and acts together to
1993 et al., Haupt 1997 et al., Honda 1997
ubiquitinate H2A and H2AX. However,
et al. and Kubbutat et al. 1997). After
RNF8 does not sustain the ubiquitylation of
phosphorylation of p53, the binding affinity is
the
the
reduced and p53 is stabilized. Stabilized
H2A by UBC13/RNF8
p53 acts as a transcription factor and can
creates a docking site for UBC13/RNF168
stimulate transcription of genes important in
which specifically polyubiquitylates H2A and
the checkpoint response. The major target
γH2AX (Stewart et al. 2009 and Doil et al.
gene is p21 that binds and inactivate
2009). These ubiquitin chains are in turn
cyclin/CDK complexes to allow a cell cycle
required for the recruitment of the BRCA1
arrest (Giono and et al. 2007). Chk2 also
complex (consisting of BRCA1, RAP80,
inhibits
Abraxas and BRCC36) and 53BP1 which
activates the Wee1 kinase which inhibits
are important checkpoint proteins (Fig. 3B).
Cdk activity directly (Sanchez et al. 1997,
Two ubiquitin-interaction motifs on RAP80
Mailand et al. 2002 and Branzei and Foiani
facilitate the accumulation of the BRCA1
et al. 2008) (Fig. 3E).
which
histones.
It
ubiquitylation of
recognizes
was
shown
that
the
Cdc25
phosphatase
and
complex to the site of DNA damage through
interaction with polyubiquitinated H2A and
Activation of Chk1
H2AX (Wang et al. 2007). How this
After the resection of the double strand
ubiquitylation regulates the recruitment of
break which is dependent on CDK activity
(Wohlbold and Fisher et al. 2009), the single
sufficient
stranded DNA is coated with replication
recovery. Many proteins are phosphorylated
protein A (RPA) which is required for the
by ATM/ATR and therefore it was likely that
recruitment of the ATR-ATRIP complex (Zou
a phosphatase was involved in checkpoint
et al. 2003) (Fig. 3C). Full ATR activation
recovery. Indeed, a phosphatase, called
requires TopBP1 binding which is recruited
Wip1, was identified to dephosphorylate
to RPA in an ATRIP dependent manner
ATM and ATR targets including ATM/ATR,
(Kumagai et al. 2006). Activated ATR
Chk1/2 and p53 (Lu et al. 2008 and
phosphorylates
recruits
Lindqvist et al. 2009). Wip1 also activates
Claspin, which in turn is essential to activate
MDM2 to target p53 for destruction by the
the protein kinase Chk1 (Wang et al. 2006)
proteasome (Zhang et al. 2009).
Rad17,
which
(Fig. 3D). Chk1 and Chk2 share many of
their substrates (Fig. 3E).
Checkpoint recovery
A critical function of the DNA damage
checkpoint is to halt cell cycle progression
until the damage is properly repaired. After
the DNA is repaired, cells resume cell cycle
progression,
also
called
checkpoint
recovery. When the DNA cannot be repaired
cells go in senescence or die by apoptosis.
A major player in DNA checkpoint recovery
is Plk1 kinase, which is phosphorylated by
Aurora A, complexed with Bora (Macurek et
al. 2008) (Fig. 3F). This phosphorylation is
required for its full activation. After its
activation, Plk1 phosphorylates the inhibitors
of CDC25; Claspin and Wee1 (Mamely et al.
2006, Watanabe et al. 2004, Mailand et al.
2006, Peschiaroli et al. 2006, van Vugt et al.
2010, Smits et al. 2002 and ToyoshimaMorimoto
et
al.
2002).
These
phosphorylations result in Claspin and
Wee1 degradation, leading to reactivation of
the CDC25 phosphatase. CDC25 can in turn
activate CDK/Cyclin complexes to allow cell
cycle progression. Plk1 also inhibits Chk2
and 53BP1 by direct phosphorylation (van
Vugt et al. 2010). However, Plk1 is not
to
fully
induce
checkpoint
Figure 3| Model for the DNA damage response and checkpoint recovery. (a) After a double strand break the MRN
complex recognizes the free DNA end and recruits ATM to the site of DNA damage. ATM is activated by
autophosphorylation and then first phosphorylates H2AX creating a docking site for MDC1. When MDC1 is recruited to
the site of DNA damage it is phosphorylated by CK2 which mediates the protein-protein interaction between MDC1 and
NBS1. This interaction leads to the accumulation of the MRN complex and thus ATM to the site of DNA damage. ATM is
now able to phosphorylate proximal H2AX molecules and thereby creates a positive feedback loop to create a wide
γH2AX region. (b) ATM phosphorylates MDC1 which serves a recruitment factor for the ubiquitin ligase RNF8. RNF8
together with its cofactor UBC13 act together to monoubiquitinate H2A and γH2AX. Once monoubiquinated it serves as a
docking site for UBC13/RNF168 which in turn polyubiquitinate H2A and γH2AX. These ubiquitin chains facilitates the
recruitment of the BRCA1 complex and 53BP1. ATM also phosphorylates the methyltransferase MMSET to facilitate its
binding to MDC1. When positioned MMSET methylates Histone H4 which is important for 53BP1 recruitment. (c) The
increased local concentration of ATM, the BRCA1 complex and 53BP1 allows activation of Chk2 by ATM dependent
phosphorylation. Chk2 induces a cell cycle arrest though MDM2, p53, wee1 and CDC25 which eventually leads to the
inhibition of CDK1/CyclinB. (d) Single stranded DNA is coated whit RPA which then recruit the ATR-ATRIP complex. ATR
is activated by the action of TopBP1. (e) Once activated ATR phosphorylates Claspin, which in turn activates Chk1. Chk2
and Chk1 share their substrates to create a cell cycle arrest. (f) When the damage is properly repaired the cells resume
cell cycle progression. Plk1 is activated by the Aurora A/Bora complex and acts together with Wip1 to allow cell cycle
progression by targeting Claspin, Chk1, Chk2, Wee1 and MDM2.
Cohesin is recruited to double-strand
double strand breaks were induced by HO
breaks and is required for DNA repair
nuclease expression which was under
Establishment of cohesion occurs during
control of a galactose inducible promoter.
DNA replication and is dependent on the
Scc1 and SMC1 were used as markers for
loading factors Scc2 and Scc4, and the
the cohesin complex and their binding to
establishment
DNA
factor
Eco1.
Intriguingly,
was
determined
by
chromatin
these factors are also important for DNA
immunoprecipitation (ChIP). The interacting
repair in G2 (Sjogren and Nasmyth et al.
chromosomal regions were analyzed by
2001). This raised the remarkable possibility
PCR.
that the novo cohesin loading and cohesion
accumulated near the region of the DNA
generation establishment can take place in
double strand break (Strom et al. 2004).
G2 in response to DNA damage. For the
Importantly, quantifications of chromosome
first evidence that sister chromatid cohesion
III containing a HO recognition site showed
is involved in DNA repair, chromosomes
at least a 5-fold increase of Scc1 in ~100 kb
from γ irradiated cells in G2 phase were
around the site of double stand break (Unal
separated by pulse-field gel electrophoresis
el al. 2004). Surprisingly, a 2-fold reduction
and amounts of full length chromosome 16
of Scc1 and SMC1 binding was observed in
and its shorter (unrepaired) variants were
regions of 2 kb proximal to the double strand
detected by Southern blot (Sjogren and
break, suggesting that either cohesin is not
Nasmyth et al. 2001). Importantly, yeast
loaded at the break itself or that cohesin has
strains containing the temperature-sensitive
moved away from the sites of DNA damage.
alleles; Scc1, Scc2, Eco1, SMC1 and pds5
In the absence of Scc2 and Scc4, SMC1 is
showed impaired DNA repair after switching
not accumulated in the region of DNA
temperature to 350C in G2 phase. This
damage, indicating that Scc2 and Scc4 are
interesting observation suggests that next to
indeed required for loading of cohesin also
the cohesin subunits also the known loading
in response to DNA damage.
Indeed,
Scc1
and
SMC1
are
and establishment factors; Scc2, Eco1 and
pds5 are required for efficient postreplicative
Involvement
of
checkpoint
proteins
in
double stand DNA repair. After this finding
cohesin recruitment
many studies were performed to find out
It was clear that the cohesin complex is
why this takes place and how this is
recruited to sites of DNA damage. The
regulated.
straight forward question arises how this is
exactly regulated. As describe above the
Accumulation of cohesin subunits to sites of
conserved loading factors are involved in
DNA damage
this recruitment. Interestingly, the important
The first step was to find out if the cohesin
checkpoint and repair proteins Tel1 (ATR),
subunits are recruited to the site of damage.
Mec1 (ATM), γH2AX and Mre11 are also
Therefore,
was
required for efficient cohesin recruitment to
generated with a HO recognition site and
sites of DNA damage (Strom et al. 2004,
yeast
chromosome
V
Unal el al. 2004). Because the recruitment
similar GAL-inducible promoter. Yeast stains
of cohesin requires conserved components
containing a temperature sensitive allele for
of the DNA damage response, it is a
Scc1 showed reestablishment of cohesion in
possibility that the mechanism of cohesin
G2/M phase after temperature switch to
loading in response to DNA damage is
37.50C (loss of S phase cohesion), but only
conserved.
after induction of HO endonuclease together
with the non temperature sensitive Scc1,
Generation of cohesion during DNA
indicating
damage in yeast cells
establishment can only occur in response to
Generation of local cohesion in response to
DNA damage.
that
postreplicative
cohesion
DNA damage
Cohesion between two sister chromatids is
Generation of genome wide cohesion in
generated during DNA replication by the
response to DNA damage
action of conserved proteins. Since the
Surprisingly,
depletion of the cohesion establishment
operators on chromosome I or chromosome
factors Eco1 and Pds5 showed impaired
IV while keeping the HO cleavage site on
DNA repair in G2 (Sjogren and Nasmyth et
chromosome III showed that cohesion is
al. 2001), it was important to find out
established
whether de novo cohesion is generated in
chromosomes. This indicates that genome-
response to DNA damage. To study the
wide cohesion is established in response to
reestablishment
chromatid
DNA damage in yeast. Similar studies
cohesion in response to DNA damage,
showed that Mre11 and Mec1 (ATR) are
elegant
required for the generation of genome-wide
of
double
cohesion
sister
strand
unbroken
temperature sensitive yeast strains ( Strom
contrast, Tel1 (ATM) and γH2AX deficient
et
cells showed no abnormalities in genome-
Chromosome
and
III
Unal
el
al.
wide sister chromatid cohesion (Unal et al.
operators which can bind to Tet repressor-
2007 and Strom et al. 2007). Furthermore, a
GFP fusion proteins to visualize sister
different
chromatids cohesion after DNA damage.
temperature-sensitive
Two GFP dots indicates cohesion loss,
chromatid cohesion was generated in G2/M
whereas one GFP dot indicates cohesion
phase after treatment with γ-irradiation and
between
The
induction of the non temperature sensitive
formation of double strand breaks is induced
SMC1. Strikingly, next to Mre11 and Mec1,
by
now Tel1 deficient cells and cells expressing
expression
sister
of
marked
2007).
Tet
the
was
performed
on
Tet
cohesion after a double strand break. In
2007
were
induced
also
using
in
al.
assays
break
experiments
by
chromatids.
site
specific
HO
study showed
that
cells
in
SMC1
lines,
sister
endonuclease under control of an GAL-
non-phosphorylateble
H2A
showed
inducible promoter and HO cleavage sites
increased sister separation compared to
on chromosome III. Ectopic non temperature
control cells when arrested in mitosis (Strom
sensitive Scc1 was also under control of a
et al. 2007). A possible explanation is that
Mre11,
Mec1,
phosphorylation
Tel1
are
and
stimulates
cohesion
generation
(Heidinger-Pauli et al. 2008) (Fig. 4). Serine
formation of local cohesion in response to
83 phosphorylation is not required for S
DNA damage while Mre11 and Mec1 are in
phase induced cohesion. This indicates that
fact required for the formation of genome-
the cohesion generation in response to DNA
wide sister chromatid cohesion. How these
damage somehow differs from S phase
checkpoint
induced
unidentified
proteins
and
for
thereby
the
proteins
essential
H2A
possibly
Importantly,
overexpression of Eco1 bypasses the defect
formation of cohesion after a double stand
imposed by the expression of the non-
break is still unknown. In addition, Scc2 is
phosphorylateble
also essential for the formation of local and
Scc1 phosphorylation by Chk1 promotes
genome-wide cohesion, indicating that the
Eco1 activity only in response to DNA
formation
cohesion
damage (Unal et al. 2007; Heidinger-Pauli et
formation requires the same loading factors
al. 2009). In addition, the phospho-mimetic
as in S phase. Overall, how the loading of
mutant, Scc1S83D, suppressed the cohesion
cohesin is regulated during DNA damage
defect in cells expressing the less active
and in S phase is very poorly understood.
Eco1R222G/K223G mutant (Heidinger-Pauli et al.
postreplicative
to
cohesion.
the
of
contribute
also
Scc1,
suggesting
that
2009).
Eco1 activity may be promoted in response
to DNA damage
Eco1 may acetylate Scc1 to promote
The next question was if the establishment
cohesion generation
factor, Eco1, is also required for the
After these findings it was likely that Eco1
establishment
break
activity is essential to generate cohesion in
induced cohesion. The fist indication that
response to DNA damage. The observation
this
that
is
of
indeed
double
the
strand
case
came
from
Scc1
phosphorylation
promotes
experiments in which overexpression of
cohesion generation after DNA damage
Eco1 generates cohesion in G2/M cells in
raised the possibility that Scc1 is acetylated
the absence of a double strand break. This
in response to DNA damage. Using multiple
strongly suggests that Eco1 activity is
sequence alignments two candidate lysines,
limiting in undamaged cells and becomes
K84/K210, were identified (Fig. 5 B/C).
more active in response to DNA damage to
These were attractive candidates because
facilitate the generation of cohesion. Since
K84 was immediately adjacent to S83 and
Mec1 is required for the generation of DNA
both shared similarities in proximal amino
damage
a
acids compared to the lysines on SMC3
activity
which are also Eco1 targets in S phase. In
possibility
induced
that
cohesion,
Mec1
it
kinase
was
regulates Eco1 acetyltransferase activity.
addition,
K210
had
previously
been
Importantly, it was shown that Chk1, which
described as an Eco1 target, but at the time
is a downstream target of Mec1, can
no phenotype was observed upon mutation
phosphorylate serine 83 of Scc1 and
of this lysine. Importantly, substitutions of
these lysines to arginines in Scc1 do not
inhibitor protein Wapl (Fig. 4A). Since lysine
affect the generation of S phase cohesion.
83 residue of Scc1 is in close proximity to
K210R
the acetylation sites of SMC3, it was likely
mutants showed a partial defect in DNA
that also the acetylation of lysine 83
damage induced cohesion and when both
promotes cohesion generation in response
residues are mutated, cohesion generation
to DNA damage by antagonizing Wapl.
is
Indeed, Wapl depleted cells allow cohesion
However, the single Scc1
completely
K84R
abolished.
and Scc1
Interestingly,
cohesin containing the Scc1K84R/K210R mutant
generation
is still enriched around the site of a double
absence of a DNA double strand break,
strand break, indicating that cohesin is
indicating that Wapl also is an inhibitor of
recruited to the site of DNA damage but
cohesion generation in G2/M phase. In
cannot
addition,
covert
into
a
cohesive
state
in
either
cells
the
presence
expressing
the
or
non-
(Heidinger-Pauli et al. 2009). The same
acetylatable Scc1 mutant which normally
phenotype
cells
blocked cohesion generation during G2/M
containing the Scc1S83A allele or Eco1
can establish cohesion in the absence of
deficient cells. Moreover, cells expressing
Wapl (Fig 4C). This suggests that Eco1
the acetyl-mimics of Scc1-K84 and Scc1-
dependent acetylation promotes cohesion
K210 can generate cohesion in G2/M in the
generation in response to DNA damage by
absence
antagonizing
is
of
also
observed
Eco1
activity
or
in
in
cells
Wapl.
Importantly,
Scc1
expressing the Scc1S83A mutant (Heidinger-
phosphorylation
Pauli et al. 2009). Furthermore, cohesion
acetylation on lysine 84 and 210 is not
generation in response to DNA damage is
required for the generation of S phase
blocked
the
cohesion. In addition, lysine 112 and 113 of
Scc1S83D/K84R/K210R mutants. All these data
SMC3 which are essential for cohesion
strongly
mediated
generation during S- phase are not required
phosphorylation of Scc1 on serine 83
in response to DNA damage. Also the
enhances Eco1 mediated acetylation of
acetyl- mimetic mutant of SMC3 cannot
Scc1 on lysine 84 and lysine 210. However,
bypass the need for DNA damage for
till now there is no biochemical evidence
cohesion
that these residues are actually acetylated in
indicating that SMC3 acetylation cannot
vivo. Mass spectrometry analysis can be
antagonize Wapl in G2/M phase (Fig. 4).
in
cells
suggests
that
expressing
Chk1
performed to test the existence of this
acetylation.
Scc1 acetylation may promote cohesion
generation in response to DNA damage by
antagonizing Wapl
In S phase, SMC3 is acetylated by Eco1
and thereby antagonizes the cohesion
on
generation
serine
in
83
G2/M
and
phase,
Figure 4| Generation of cohesion during DNA damage. (a) During S-phase SMC3 (red) is acetylated by EcoI,
which antagonizes Wapl to allow cohesion generation or maintenance. (b) In response to DNA damage a wide
γH2AX region around the DNA double strand break is formed and the cohesin complex is recruited via Scc2 and
Scc4 by a still unidentified mechanism (indicated by arrowheads). Cohesion generation is inhibited by Wapl activity.
(c) Mec1 (ATR) activates Chk1, which in turn phosphorylates Scc1 presumably to recruit Eco1 to the site of DNA
damage. Eco1 may acetylate Scc1 to promote cohesion generation by antagonizing Wapl.
Post-translational
of
not (Fig. 5A/B/C). This suggests that the
cohesin subunits in response to DNA
establishment of cohesion in response to
damage in human cells
DNA damage may differ between these two
Generation of cohesion is apparently not
species.
strictly limited to S phase, but can also
quantitative
reestablish in G2 in response to DNA
spectrometry analysis showed that SMC3
damage.
acetylation
acetylation at lysine 105 and lysine 106 is
antagonizes Wapl during DNA replication in
induced in response to DNA damage in
S phase and in response to DNA damage
human cells (Beom-Jun et al. 2010). This
by targeting SMC3 or Scc1 respectively in
acetylation is dependent on ESCO1 and on
yeast cells. While the acetylation sites on
ATM/ATR
SMC3 are conserved between yeast and
phospho-proteomic
human,
ESCO1
Eco1
modifications
dependent
remarkably
the
putative
Scc1
acetylation and phosphorylation sites are
Indeed,
recent
studies
SILAC-based
kinase
as
a
activity.
mass
Recently,
screen
novel
using
ATM
a
identified
substrate,
suggesting that ESCO1 may be under
control of ATM kinase activity (Matsuoka et
al. 2007). However, ESCO1 could also be
an indirect target of ATM, because the
phospho-proteomic screen was performed
using ATM inhibitors.
How
and
whether
ESCO1
is
recruited to the site of DNA damage and
how it is further regulated remains unknown.
Interestingly, using ChIP-sequencing it was
shown that postreplicative binding of SMC1
and SMC3 was not limited to the DNA
damaged sites but was observed genomewide on pre-existing cohesin binding sites.
In addition, when serine 1083 of SMC3 (a
known
ATM
site,
discussed
later)
is
substituted to alanine, cohesin binding is
impaired. How these post translational
modifications enhance cohesin binding to
chromatin
is
unclear.
Because
these
modifications are in close proximity to the
ATPase domain it is a possibility that these
modifications
regulate
SMC3
ATPase
activity. A second likely possibility is that
these
modifications
modulate
the
antiestablishment factor Wapl as in S phase.
Figure 5| Comparison of phosphorylated and
acetylated regions of SMC3 and Scc1 between
yeast and human. (a) Eco1 dependent acetylation
An important note is that the generation of
sites on SMC3. (b) Chk1 dependent phosphorylation
cohesion in response to DNA damage was
and Eco1 dependent acetylation site on Scc1. (c) Eco1
thus far not been shown in human cells. In
dependent acetylation site on Scc1.
conclusion
different
post
translational
modifications appear to regulate the loading
of the cohesin complex and generation of
cohesion in response to DNA damage.
Remarkably these modified residues are not
all conserved between yeast and human.
SMC1 is a target for ATM in response to
343 to activate the S phase checkpoint.
DNA damage in human cells
Importantly, in cells expressing NBS1 in
As mentioned earlier, ATM is the major
which serine 278 and 343 are mutated to
kinase that regulates cell cycle checkpoints,
alanines, the phosphorylation of SMC1 on
DNA
serine
repair
and
recombination.
This
957
was
abolished
and
suggests the possibility that also the cohesin
phosphorylation of serine 966 was reduced
subunits are under the control of this master
(Yazi et al. 2002). This suggest that the
regulator in response to DNA damage.
phosphorylation of NBS1 acts upstream of
Indeed, in human cells, SMC1 was found to
SMC1
be phosphorylated in vivo and in vitro in an
mechanism
ATM
Mass
(discussed later). BRCA1 mutants who can
spectrometry analysis identified that both
not be phosphorylated by ATM do not affect
serine
SMC1
dependent
957
and
manner.
966
of
SMC1
are
phosphorylation.
remains
to
The
be
phosphorylation.
exact
established
However,
Co-
phosphorylated by ATM in vivo upon
immunoprecipitation experiments showed
ionizing
that
irradiation
(Kim
et
al.
2002).
SMC1 can interact
with BRCA1,
However, in ATM deficient cells these
indicating that SMC1 and BRCA1 may form
residues were still phosphorylated after
a complex in response to DNA damage
exposure to ultraviolet light or when DNA
(Yazi et al. 2002). The function of this
replication is inhibited by hydroxyurea. This
complex and whether or not this complex is
indicates that an additional kinase(s) can
DNA
phosphorylate SMC1 in response to DNA
unknown.
damage-regulated
is
currently
damage or a blockade of replication. Indeed,
it
was
described
that
that
SMC1
is
SMC1
phosphorylation
is
important
to
phosphorylated by ATR in ATM deficient cell
maintain the S phase and the G2/M
lines
checkpoint
in
response
to
DNA
damage
(Tomimatsu et al. 2009).
It
became
evident
that
SMC1
phosphorylation is involved in the S phase
SMC1 phosphorylation of serine 957 and
checkpoint since the non- phosphorylateble
966 are NBS1 and BRCA1 dependent
SMC1
Many proteins and protein complexes are
increased DNA synthesis in response to
involved in the DNA damage response.
DNA damage, compared to wild type SMC1
Early studies showed that phosphorylation
expressing cells (Yazi et al. 2002). In
of serines 957 and 966 were abolished in
addition,
cells lacking NBS1 or BRCA1, indicating
metaphase
that these phosphorylation events are NBS1
increase of chromosomal aberrations in
and BRCA1 dependent (Kim et al. 2002;
cells expressing SMC1S957A/S966A, suggesting
Yazi et al. 2002). NBS1 is part of the MRN
that these phosphorylations are important
(MRE11/RAD50/NBS1)
is
for either sensing the DNA damage , or for
phosphorylated by ATM on serine 278 and
maintaining the G2/M checkpoint (Kitagawa
complex
and
mutant
(SMC1S957A/S966A)
chromosome
arrested
cells
showed
spreads
showed
of
an
et
al.
2004).
these
(Tomimatsu et al. 2009). How SMC1 is
phosphorylations are also important for DNA
phosphorylated in response to hypoxia was
repair is unknown. Interestingly, antibodies
unclear. In cells treated with the hypoxia-
detecting
co-
mimetic treatment, Desferrioxamine (DFO),
NBS1,
SMC1 is phosphorylated at serine 966 in an
53BP1, CHK2 and BRCA1 foci after ionizing
ATM independent manner. Importantly, in
irradiation,
is
ATR deficient cells, the levels of SMC1,
phosphorylated at the site of DNA damage.
Chk1 and NBS1 phosphorylation greatly
Importantly, cells expressing SMC1S957A/S966A
decreases, indicating that these proteins are
showed no aberration in localization of these
phosphorylated by ATR. However, these
ATM substrates,
SMC1
phosphorylations could also be indirect. In
phosphorylation events is not involved in
addition, DNA-PKcs deficient cells showed
recruiting these ATM substrates (Kitagawa
no abnormalities in phosphorylation of these
et al. 2004). However, probably other
proteins. Importantly, Chk2 phosphorylation
regions of SMC1 or other cohesin subuntis
was not affected in ATR deficient cells, but
are involved in the recruitment of important
was reduced in cells lacking DNA-PKcs.
checkpoint proteins to the site of DNA
This suggests that two different pathways
damage (discussed later).
are activated in response to DFO. First,
pS957
localization
with
Whether
SMC1
ATM,
suggesting
showed
γH2AX,
that
SMC1
indicating
that
SMC1, NBS and Chk1 are phosphorylated
The role of SMC1 in hypoxia induced
in an ATR dependent manner, whereas
apoptosis
Chk2
DNA damage signaling pathways are also
dependent. In addition, ATR deficient cells
initiated in response to hypoxia, including
show
phosphorylation of SMC1 on serine 966.
exposure
Hypoxia is mainly observed in solid tumors
expressing SMC1 in which serine 966 was
due the reduced availability of oxygen
mutated to alanine also show a reduction of
supply. The lack of oxygen can induce
apoptosis after exposure of DFO. This
aberrant DNA repair which will lead to
suggest that ATR mediated phosphorylation
genetic
of SMC1 is responsible for this phenotype.
instability.
Hypoxia
induced
activation of ATM and ATR is independent
of the MRN complex (Bencokova et al.
2009).
As
describe
phosphorylated
in
an
above,
ATM
SMC1
is
dependent
manner in response to ionizing radiation, but
ATM is dispensable after treatment with
hydroxyurea or UV irradiation. Recently it
was found that in ATM deficient cells SMC1,
p53,
Chk1,
Chk2
and
53BP1
are
phosphorylated in a ATR dependent manner
phosphorylation
a
reduction
to
DFO.
of
is
DNA-PKcs
apoptosis
after
Importantly,
cells
The role of cohesin in the DNA damage
damage induced checkpoint signaling. In
induced G1/S and G2/M checkpoint in
addition, when the checkpoint was switched
human cells
off after one hour of induction of DNA
Besides the role in DNA repair, cohesin is
damage, control cells showed less broken
also involved in the DNA damage induced
chromosomes than the sororin depleted
checkpoint activation. As describe above,
cells, indicating that indeed also in human
SMC1 is phosphorylated on serine 957 and
cells, cohesion is essential for efficient DNA
966 in an ATM dependent manner upon γ-
repair. However, this finding needs to be
irradiation.
phosphorylation
confirmed by measuring checkpoint activity,
accumulates at irradiation induced foci in the
for instance by determination of p21 levels
nucleus
in G2 cells lacking Scc1 or sororin.
SMC1
and
cells
expressing
non-
phosphorylateble mutants shows defect in
the intra-S phase and the G2/M checkpoint
The cohesin complex regulates Chk2 kinase
checkpoint (Yazi et al. 2002 and Kitagawa et
activity
al. 2004). Likewise, residues of SMC3
Interestingly, semi-quantitative immunoblot
(serine 1067 and 1083) are phosphorylated
and immunofluorescence analysis showed
by
that
ATM
and
expression
non-
the
phosphorylation
of
Chk2
on
phosphorylateble mutants also affects the
threonine 68, which is essential for its
intra-S phase checkpoint.
activation, is reduced in Scc1 and SMC3
In human cells sororin is required for
depleted
cells.
Remarkably,
the
DNA repair in G2 phase (Schmitz et al.
phosphorylation of ATM, Chk1 and γH2AX
2007). In sororin depleted cells, cohesin can
was increased in Scc1 and SMC3 depleted
associate with chromatin, but is unable to
cells during and before DNA damage. A
generate
possible explanation
or
maintain
sister
chromatid
could be that in
cohesion. The role of cohesin in the G2/M
cohesin deficient cells, DNA double strand
checkpoint became evident when double
breaks accumulate due to defects in DNA
strand breaks were induced in Hela cells by
repair. Importantly, sororin is not required for
γ-irradiation or treatment with etoposide in
Chk2 activation, suggesting that cohesin
G2 phase. To determine the activity of the
and not its ability to mediate cohesion is
G2/M checkpoint, chromosomal spreads of
essential for Chk2 activation. In addition,
mitotic chromosomes were analyzed for
cohesin is also required to active Chk2 in
broken chromosomes (Watrin et al. 2009).
G1 phase, again indicating that this process
In these experiments, Scc1 and SMC3
is independent of the generation of sister
depleted cells overrides the G2/M DNA
chromatid cohesion.
damage checkpoint and enters mitosis with
broken chromosomes. Importantly, sororin is
The cohesin complex recruits important
not required for the G2/M checkpoint,
checkpoint proteins to sites of DNA damage
indicating that cohesin and not its ability to
After irradiation, several proteins which are
generate cohesion is essential in DNA
involved in the DNA damage response are
recruited to the site of DNA damage.
contributed to the formation of cohesion is
Immunofluoresce experiments showed that
currently
Scc1 and sororin are not involved in the
recruitment
enrichment of Mre11, Nbs1, MDC1, γH2AX
cohesion in G2/M phase are dependent on
and ATM-S1981P to the site of DNA
the conserved loading factors Scc2 and
damage. However, MDC1 and γH2AX are
Scc4. How these factors recruit cohesin to
more diffuse over the chromatin in Scc1
chromatin remains to be investigated, both
depleted
in S phase and in response to DNA
cells.
In
contrast,
proper
localization of the mediator protein 53BP1 is
unclear.
and
Intriguingly,
the
the
establishment
of
damage.
strongly dependent on Scc1 but not on
sororin. Since 53BP1 is known to be
Yeast
required for Chk2 phosphorylation, it is
phosphorylation by Chk1 promotes Eco1
possible
dependent
that
cohesin
mediates
Chk2
studies
argue
acetylation
that
and
Scc1
thereby
activation by promoting the recruitment of
antagonizes the cohesion inhibitor Wapl.
53BP1 to the sites of DNA damage. It is an
However, the serine and lysine residues in
interesting possibility that cohesin is a
Scc1 are not conserved between yeast and
crucial recruitment factor for more essential
humans, indicating that in this respect the
checkpoint
is
establishment of DNA damage induced
important to look for interaction partners of
cohesion may differ between these species.
the cohesin subunits specifically in response
Importantly, human studies showed that
to
Immunoprecipitation
lysine 105 and lysine 106 of SMC3 are
experiments in combination with mass
acetylated not only at the site of DNA
spectrometry analysis could help to identify
damage but also in a genome wide fashion.
such interaction partners.
These
proteins.
DNA
Therefore,
damage.
it
acetylations
are
dependent
on
ESCO1 and on ATM/ATR. Whether or not
Concluding
remarks
and
future
perspectives
Above,
the
this contributes to the establishment of DNA
damage induced cohesion is unknown.
role
the
Since important checkpoint proteins are
generation of cohesion in response to DNA
involved in the loading of cohesin and
damage
studies
generation of cohesion in response to DNA
became clear that cohesin subunits are
damage it is possible that cohesin loading
enriched around the site of DNA damage
and cohesion generation may differ from S
and are able to transform into the cohesive
phase. Interestingly, phospho proteomics
state.
wide
revealed ESCO1 as a potential target for
cohesion is established. To allow cohesion
ATM, raising the possibility that ESCO1
establishment after DNA replication, the
activity is under control of ATM kinase
important checkpoint proteins Tel1 (ATM),
activity.
Mec1
ESCO1 phosphorylation increases its local
are
of
described.
Importantly,
(ATR),
required.
How
cohesin
Yeast
also
Chk1
these
and
genome
and
MRE11
proteins
are
exactly
One possibility could be that
recruitment to the chromatin in response to
expressing the non phosphorylateble mutant
DNA damage.
of NBS1. BRCA1 phosphorylation does not
affect
the
phosphorylation
co
of
SMC1.
Thus, in yeast and possibly in mammalian
However,
cells local and genome wide cohesion is
experiment showed that BRCA1 interacts to
generated. However, the actual function of
SMC1. Which domains are involved and the
the generation of cohesion in response to
function
DNA damage is still under debate. A straight
unknown.
of
immunoprecipitation
this
complex
is
currently
forward model for local cohesion is to bring
the DNA strands in close proximity to allow
SMC1 phosphorylation is involved in the S
proper DNA repair. But why does DNA
phase
damage trigger genome wide cohesion
Importantly,
establishment? One explanation is to inhibit
localized at the sites of DNA damage.
gene transcription to avoid unregulated
However, these phosphorylation events do
signaling
important
not affect the localization of the checkpoint
regulatory proteins are mutated due DNA
proteins NBS1, 53BP1, CHK2 and BRCA1.
damage.
In contrast, 53BP1 localization is dependent
pathways
when
and
the
G2/M
checkpoint.
phosphorylated
SMC1
is
on the cohesin complex, indicating that
In human cells it is shown that SMC1 is
other regions on SMC1 or other subunits of
phosphorylated on serine 957 and 966 by
the complex are involved in the recruitment
ATM in response to DNA damage or ATR
of 53BP1 and possibly more important
after treatment with a hypoxia-mimetic
checkpoint
treatment.
these
phosphorylation on serine 68 is reduced in
phosphorylation events were abolished in
Scc1 and SMC3 depleted cells. 53BP1 is
the absence of NSB1 or BRCA1, suggesting
required for
that SMC1 phosphorylation is downstream
interesting possibility could be that the
of these checkpoint proteins. Further studies
cohesin
showed that NBS1 phosphorylation by ATM
recruitment
promotes SMC1 phosphorylation. The exact
phosphorylation. In addition, a more diffuse
regulatory mechanism remains unclear. A
localization
possible explanation could be that NBS1
observed in cells lacking Scc1. Interestingly,
phosphorylation
cohesin
in an artificial situation in which the cohesin
complex to allow local phosphorylation of
complex cannot transfer into its cohesive
SMC1.
is
state, 53BP1 does localize properly. So far,
phosphorylated in an ATM/ATR dependent
it is unknown how the cohesin complex
manner to regulate the S phase checkpoint
recruits 53BP1 or other novel important
after irradiation. Until now no studies were
checkpoint
performed on the localization of SMC1 or
immunoprecipitation and proteomics studies
SMC3 in cells lacking NBS1 or cells
need
In
Importantly,
recruits
addition,
the
also
SMC3
to
proteins.
Interestingly,
Chk2
Chk2 phosphorylation.
complex
and
of
facilitates
thereby
MDC1
proteins.
be
performed
53BP1
allows
and
Chk2
γH2AX
Large
to
An
allow
is
scale
the
identification of potential interaction partners
This could then lead to the removal of
of the cohesin complex and eventually its
cohesin in a similar manner as during the
post-translational modifications in response
prophase-pathway.
to DNA damage. This method also has the
potential to identify unknown proteins that
The
crucial
residues
and
their
post
are important for the recruitment of the
translational modifications that are important
cohesin complex to chromatin.
for generation of cohesion in yeast are not
all conserved in mammalian cells. Therefore
During checkpoint recovery, ATM and ATR
it is important to identify and characterize
substrates
or
the key regulatory factors that are essential
degraded to allow cell cycle resumption.
for DNA damage induced cohesion loading
Whether
are
and cohesion establishment in human cells.
dephosphorylated or degraded is currently
Further work is required to investigate the
unknown. It could be simply that Wip1
role of the cohesin complex in response to
dephosphorylates SMC1 and SMC3 during
DNA
checkpoint recovery. Second, Plk1 kinase
understand the exact roles of local and
activity is high during checkpoint recovery
genome wide cohesion generation after
which may lead to phosphorylation of Scc3.
DNA damage.
are
SMC1
dephosphorylated
and
SMC3
damage.
This
is
essential
to
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