Lecture 3:
Origins and Replication Initiation
Analyzing role and function of sequence elements (sequence specific assays)
Regulation through feedback inhibition by product
5’
3’
5’
5’
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3’
Lecture 4:
Eukaryotic Initiation and Regulation
In vivo analysis of protein interactions and complex assembly
Increasing the power of genetic tools with better molecular phenotypes
3’
5’
A Tale of Two Systems
E. Coli oriC
S. cerevisiae ARS
Develop in vitro system
Genetically identify
initiation factors
Establish “purified” system
Localize factors to origins
and/or replication forks
Create partial reactions and
structurally analyze intermediates
Establish order of
assembly during initiation
and cell cycle progression
Infer protein function and
develop specific assays
Develop in vitro system
and specific assays
Future mechanistic studies
(great Bioreg proposals)
Prokaryotic and Eukaryotic Replication Initiation Activities
5’
5’
3’
3’
3’
3’
5’
5’
5’
3’
Converting DS DNA to replication fork
E. coli
S. cerevisiae
1. Recognize initiation site (replication origin)
DnaA binds oriC
ORC binds origins
2. Expose single-stranded templates (unwind)
DnaA
ORC? Mcm2-7?
DnaC loads DnaB
Cdc6, Cdt1 load Mcm2-7
Sld2, Sld3, Dpb11 load
Cdc45 & GINS
Primase
DNA Pola - primase
3. Load helicase at nascent fork
4. Prime DNA synthesis
5. Load polymerase(s)
Sld2, Sld3, Dpb11 loads
DnaB binds t subunit
DNA Pol  complex
SSB & primer-template
?? loads
bind Clamp-Loader & Clamp
DNA Pol  complex
2-Stage Model for Protein Assembly During Replication Initiation
M Phase
G1 Phase
S Phase
Cdc7-Dbf4
CDK
Trigger
License
GINS
Post-RC
Pre-RC
Pre-IC
Initiation
Genetic Screens Enriching for Replication Initiation Mutants
Conditional Mutants:
cell division cycle (cdc)
budded morphology
1N DNA content
execution point before elongation
Initiation or Elongation?: Execution Point Analysis
Requires independent and reversible means of inactivating two functions plus an “endpoint” assay
A mutated initiation function is completed by the time elongation is blocked
Initiation
Elongation
1st shift HU
2nd shift ts
Cell Cycle
Completed
A mutated elongation function is still needed when elongation blocked
Initiation
Elongation
1st shift HU
2nd shift ts
Cell Cycle
Remains Blocked
HU = hydroxyurea which blocks replication elongation by inhibiting dNTPs biosynthesis
Genetic Screens Enriching for Replication Initiation Mutants
Conditional Mutants:
cell division cycle (cdc)
budded morphology
1N DNA content
execution point before elongation
cdc6
cdc46/mcm5
cdc47/mcm7
cdc54/mcm4
cdc7
dbf4
cdc45
Genetic Screens Enriching for Replication Initiation Mutants
Conditional Mutants:
cell division cycle (cdc)
Hypomorphic Mutants:
minichromosome maintenance (mcm)
% cells containing plasmid
WITH selection
% cells containing plasmid
withOUT selection
budded morphology
1N DNA content
faster loss of minichromosome (I.e.
selectable plasmid) from population
execution point before elongation
suppression of mcm phenotype with
multiple plasmid origins
cdc6
cdc46/mcm5
cdc47/mcm7
cdc54/mcm4
cdc7
dbf4
cdc45
cdc6
mcm2
mcm3
mcm5/cdc46
mcm10
Gathering More Suspects: Guilt by Association
genetic & physical interactions with replication genes/proteins
Example: How Araki found many of the genes required for triggering initiation
POL2
high copy
suppression
DPB2
DNA Pol 
subunits
synthetic
lethality
DPB11
SLD1 = DPB3
Loaders for Pol 
Cdc45 & GINS
SLD2
SLD3
SLD4 = CDC45
PSF1
SLD5
coIP & mass spec
GINS complex
PSF2
PSF3
CMG helicase
holoenzyme subunits
S. cerevisiae origin: ~120 bp ARS1
A B1
B2
B3
A is an essential ARS consensus sequence (ACS)
B1, B2, & B3 partially redundant (linker scan)
Yeast Origin Recognition Complex (yORC): A 6 Subunit Initiator
Biochem: Binding Activity
ARS1 Footprint
Genetics: Hint from Mutational Correlation
ARS
mutations
Poor in vitro binding activity
Poor in vivo origin function
Genetics: Establishing Initiation Function
2-D gel analysis of ARS1 initiation
Note: most other eukaryotic ORCs do
NOT have such sequence specificity
ORC5
orc5-1
In Vivo Assays for Protein DNA Interactions
Identifying intermediates in the assembly of initiation complexes on DNA
Genomic Footprint
Genomic
Footprint
DNA:yORC
Preferred binding sites
of specific proteins
yORC1
ChIP preIP
ARS1
DNA
Protein binding and/or
distortion of specific sites
Chromatin IP (ChIP)
control
control
Gel
ARS305
control
Microarray
yORC1 ChIP-chip (chromosome VI)
Pre-Replicative Complex (pre-RC) in G1 Phase
Temporal analysis of genomic footprint at origins
Yeast 2µ origin
M G1 S-G2-M
ORC hypersensitive site
reduced in G1 phase
Extended protection
of B domainIn G1 phase
Speculation: ORC binds origin throughout the
cell cycle and is joined by other proteins in G1
phase to “license” origins for initiation
Ordered Assembly of Proteins at Origins During G1 & S
Using ChIP to establish temporal order and genetic
dependencies of proteins assembling at the origin
Example: G1-specific recruitment of Mcm7 is dependent on Cdc6
Synchronized
yeast culture
- Cdc6 or + Cdc6
G1
S
G2
M
G1
time points sampled for Mcm7 ChIP
- Cdc6
G1
S - G2 -M
+ Cdc6
G1
S - G2 -M
preIP
control
control
ARS1
control
Using both Biochemistry and Genetics to understand function
Dynamic Protein Associations Through G1 and S
Combining temporal and spatial analysis of replication and binding in synchronized cells
BrdU incorporation monitors fork movement
Cdc45 ChIP-chip tracks with fork movement
Some replication proteins that load at origins later move with the forks:
Mcm2-7, Cdc45, GINS, Mcm10, Dpb11, DNA Pol a, DNA Pol , DNA Pol , PCNA
(clamp), RFC1-5 (clamp loaders), RFA
2-Stage Model for Protein Assembly During Replication Initiation
M Phase
G1 Phase
S Phase
Cdc7-Dbf4
CDK
Trigger
License
GINS
Post-RC
Pre-RC
Pre-IC
Initiation
Biochemical insights into Mcm loading and activation
Pre-RC Assembly (helicase loading)
Can control addition order of protein, cofactors, or inhibitors
Can substitute mutant/modified proteins with altered activities
Can analyze structures with greater resolution and accuracy
Mcm2-7 doublehexamer remains
on DNA after high salt wash
ORC-DNA
ATP
Helicase Activity
Drosophila extract
purify
helicase
activity
Cdc45 - Mcm2-7 - GINS
(CMG - helicase “holoenzyme”)
Cdc6
Cdt1-Mcm2-7
EM reconstruction
side
end
Replication Elongation Blocking
C
N
hexamer
N
C
hexamer
Discussion Paper
2-Stage Model for Protein Assembly During Replication Initiation
M Phase
G1 Phase
S Phase
Cdc7-Dbf4
License
core helicase
loaded around
DS DNA
CDK
Trigger
helicase holoenzyme
loaded around
unwound SS DNA
GINS
Post-RC
Pre-RC
Pre-IC
Initiation
A Simplified View of Cyclin Dependent Kinases (CDKs)
CDK regulation
CDKs = kinase + cyclin
cyclins undergo periodic synthesis and proteolysis
different cyclins activate CDKs to promote
different cell cycle events
G1 cyclins
not shown
CDK in S. cerevisiae
kinase = Cdc28
G1 cyclins = Cln1-3
S & M cyclins = Clb1-6
Molecular Biology of the Cell, 4th Ed.
Activation of CDKs and DDKs in S phase trigger origin initiation
S
Dbf4-Cdc7
(DDK)
Post-RC
Pre-RC
Clb-Cdc28
(CDK)
Pre-IC
Initiation
G2
Identifying CDK and DDK targets for replication initiation
How to identify in vivo targets of kinase?
kinase substrate in vitro
1) Kinase substrate in vivo
phosphorylated in vivo in kinase dependent manner
in vitro and in vivo phosphorylation sites overlap
2) (Necessity) Phosphorylated sites essential for kinase function
3) (Sufficiency??) Phosphomimic mutations allow bypass of kinase requirement
Sld2 and Sld3 are essential replication targets for CDK triggering of initiation
Sld2 and Sld3 phosphorylation promotes their independent binding to Dpb11
Sld2 phosphorylation promotes formation of “pre-Loading Complex” with GINS, pol , Dpb11
DDK
Mcm4 and Mcm6 are essential targets for Cdc7-Dbf4 kinase (DDK)
A temporal program regulates DNA replication within S phase
Locate earliest DNA synthesis
One example: Microarray analysis of copy number
Earlier Initiation
Later Initiation
Temporal control of DNA replication through earlier DDK action?
S
G2
CDK
DDK
Sld3
Cdc45
Early
Origins
Pre-RC (DDK activated)
Initiation
Post-RC
DDK CDK
Post-RC
Late
Origins
Pre-RC
Pre-RC
Initiation
What distinguishes earlier from later origins?
What determines when a later origin becomes ready to fire?
Why is there temporal control of DNA replication within S phase?
Cell cycle control of origin function must be highly efficient
X
X
X
X
CDK
if you want a 50,000 origin genome to NOT re-initiate with 99.5% fidelity
then re-initiation at each origin must be prevented with 99.99999% fidelity
50,000
(.9999999)
= .995
X
X
The CDK paradigm for once and only once replication
G2
M
G1
MCM
2-7
a ssembl e
pr e-RC Cdc6 Cdt1
ORC
S
Start
tri gger
i ni tia tion
G2
ORC
ORC
ORC
Fork
preRC assembly
ORC
Fork
ORC
NO preRC assembly
ORC
Cdc6
MCM
2-7
Cdt1
CDK
NO
triggering initiation
Sld2
Sld3
trigger initiation
M
G1
The CDK paradigm for once and only once replication
G2
M
G1
MCM
2-7
a ssembl e
pr e-RC Cdc6 Cdt1
ORC
S
Start
G2
MCM
2-7
Cdc6 Cdt1
tri gger
i ni tia tion
Fork
ORC
preRC assembly
ORC
ORC
Fork
ORC
Some preRC re-assembly
ORC
X
Cdc6
MCM
2-7
X X
Cdt1
X
CDK
NO
triggering initiation
Sld2
G1
ORC
ORC
ORC
Fork
M
Sld3
trigger initiation
Fork
CDKs Target Multiple Proteins to Block pre-RC Re-assembly
Overlapping mechanisms ensure re-initiation is blocked at thousands of origins
In budding yeast, CDK phosphorylation of
1) Mcm3 promotes Mcm2-7 nuclear exclusion
2) Cdc6 promote its proteolysis
3) Cdc6 promotes CDK binding and inhibition
4) Orc2/Orc6 inhibits recruitment of Cdt1-Mcm2-7
5) CDK binding to Orc6 inhibits ORC function
The extensive overlap of mechanisms is conserved, NOT specific mechanisms
Metazoans have additional CDK-independent mechanisms inhibiting re-initiation
Cell cycle control of origin function must be highly efficient
X
X
X
X
CDK
if you want a 50,000 origin genome to NOT re-initiate with 99.5% fidelity
then re-initiation at each origin must be prevented with 99.99999% fidelity
50,000
(.9999999)
= .995
X
X
How important is it to prevent re-initiation?
Partial loss of replication control in yeast can greatly induce genomic instability
Gene Amplification
Aneuploidy
Other Instability?
Translocations?
Inversions?
Loss of Heterozgosity?
Beyond Initiation: Keeping the Fork Going Through Thick and Thin
Many genomic insults are now thought to originate from replication accidents
DNA lesions induce responses to: (1) protect stalled forks
(2) bypass lesions
(3) delay further initiation
(4) block cell cycle
QuickTime™ and a
decompressor
are needed to see this picture.
2
3
1
4
Segurado & Tercero, Biol. Cell (2009) 11:617-627
Expanding influence of replication on other processes
Epigenetic Chromatin States
-- how are chromatin states inherited during DNA replication?
-- does replication timing contribute to this inheritance?
Development and Differentiation
-- do replication timing changes help execute developmental decisions?
Sister Chromatid Cohesion
-- how is the establishment of cohesion coupled to DNA replication?
Meiotic Recombination
-- how is initiation of DS breaks coupled to DNA replication?
Cancer Biology
-- does loss of replication control contribute to oncogenic genomic instability?
Evolution
-- does sporadic re-replication contribute to genetic variation?
The Replication Checkpoint Insures Mitosis Does Not Proceed
If Replication Is Delayed or Blocked
Standard view of cell cycle
Branched view of cell cycle
elongation
termination
Replication
mec1
rad53
Mec1
Start
Rad53
Segregation
mitosis
Passage through Start independently sets into motion both Replication and Segregation
Replication is normally completed before mitosis can begin, allowing their proper temporal order
Block or delay in elongation signals checkpoint mechanism (e.g. Mec1, Rad53) to prevent mitosis
Exact nature of signal is unknown but it cannot be generated unless some initiation occurs
(abnormal fork structure? stalled replisome?)
Checkpoint Mechanisms Also Stabilize Stalled Replication Forks
Abnormal replication intermediates accumulate when replication is delayed in checkpoint mutants
Unperturbed Replication
dNTP Depleted Replication
WT
hemi-replicated
large gaps
collapsed forks
Checkpoint
Mutant
(rad 53)
Based on EM analysis by Sogo & Foiani Labs, Science (2002) 297:599
Possible functions of checkpoint mechanisms at forks during replicative “stress”
maintain proper coordination of leading and lagging strand synthesis
prevent fork collapse and branch migration
These protective functions allow forks to resume replicating when the stress is removed
Origin Usage is Regulated During Development
Developing Frog
S Phase Length
~ 10 kb interorigin distance
20-25 min
~ 20 kb interorigin distance
~ 100 -200 kb interorigin distance
~ 6-8 hrs
Line represents DS DNA
Origin Timing is Influenced by Chromatin Structure
S. cerevisiae origins fire throughout S phase in a defined and reproducible order
Position effect on origin timing
ARS501
late
Chr 5
Chr 4
Telomeric heterochromatin can delay origin firing
WT
Sir dependent heterochromatin
TEL
ARS1
early
Y’ ARS fires late
Swap positions
sir mutant
ARS1
late
Chr 5
TEL
Y’ ARS fires early
Chr 4
ARS501
early
from Brewer and Fangman labs, Cell (1992) 68:333
from Gottschling Lab, Gene & Dev (1999) 13:146
Replication is Coupled to Establishment of Sister Chromatid Cohesion
Speculative polymerase switch model
Cohesin complexes (Scc1,Scc3,Smc1,Smc3) hold sisters
together and ensure bipolar spindle attachment
Cohesins must be present on chromatin during
replication for proper cohesion to occur
Replication-like proteins have been implicated
in the establishment of cohesion:
- two DNA polymerases of the s family,
Trf4 and Trf5
s
figure from Hieter Lab, Mol Cell (2001) 7:959
- a modified RFC clamp loader with Ctf8,
Ctf18, and Dcc1 substituting for Rfc1
Duplication of Nucleosome Structure During Eukaryotic Replication
Parental nucleosomes distribute randomly to daughter DNA
New histones complete the daughter complement of nucleosomes
Model for new histone deposition at replication forks
Histone Deposition Factors (CAF-1 and/or Asf1)are recruited to
newly replicated DNA by PCNA and deposit H3H4 tetramers
H2A-H2B dimers self assemble onto H3-H4 tetramers
caf-1 asf1 double deletion mutants are viable in yeast,
suggesting other histone deposition mechanisms exist
How higher order chromatin structure is duplicated is not known
Does the replicator model apply to metazoan origin?
The ARS plasmid assay has failed to identify metazoan origins
Alternative strategy:
physically map sites of initiation
genetically identify sequences required for initiation at those sites
example: potential origin in human globin gene cluster
100 kb
LCR
E
Gg
Ag

b
IR
Deletions in either region(from thallasemia patients) inactivates the IR
IR = Initiation Region based on physical mapping of nascent strands
LCR = Locus Control Region (required for transcriptional activity of entire cluster)
Alternative hypothesis:
little sequence specificity for initiation site
zones of initiation established by chromatin structure
Eukaryotic Replication Initiation is Coupled to the Cell Cycle
Helicase(?)
Loaders
Initiator
Helicase(?)
Primase
2
1
Kinases
Polymerase
Origin Recognition
Complex
pre-Replicative Complex
(pre-RC)
Replicative Complex
(RC)
Two fundamental stages of initiation defined primarily by Chromatin IP and Chromatin Association assays
Stage 1: pre-RC assembly in G1 phase makes origins competent to initiate DNA replication
Stage 2: Passage through Start (G1 commitment point) activates Clb-Cdc28 kinase and Cdc7-Dbf4 kinase
which trigger formation of the replicative complex and initiation of DNA replication
Using both Biochemistry and Genetics to understand function
Structural Analysis of Intermediates
Examples of structural features of intermediates that can be monitored
Nucleic Acids
Proteins
Complexes
Size
Cofactor (NTP) Status
Composition
Shape
Conformation
Stoichiometry
DS versus SS
Modification
Conformation
Strand Pairing
Ligand Binding
Interacting Sequences
Modification
Covalent Linkages
Interacting Domains
Covalent Linkages
Topology
2-Stage Model for Protein Assembly During Replication Initiation
M Phase
G1 Phase
S Phase
CDK
Cdc7-Dbf4
Trigger
License
GINS
Post-RC
Pre-RC
Pre-IC
Initiation
Using both Biochemistry and Genetics to understand function
Metazoans have CDK-independent mechanisms to block re-initiation
The cell cycle control of replication must be highly efficient
X
X
X
X
Fidelity: > 99.999% block for genome with 50,000 origins
Hayles et al. Cell 1994
Cyclin dependent kinases implicated
X
X