Download Bioreg2017_Replication3_V4

Lecture 3:
Origins and Initiation and Regulation
Analyzing role and function of sequence elements
Increasing the power of genetic tools with better in vivo molecular phenotypes
Regulation through ordered assembly of protein machines
Studying the inhibition/activation of activities to uncover regulatory mechanisms
Activities needed to initiate DNA replication
5’
3’
3’
5’
5’
helicase
DNA polymerase
3’
Converting DS DNA to a replication fork
Activities needed to initiate DNA replication
5’
origin
3’
5’
3’
helicase
Converting DS DNA to a replication fork
1. Recognize initiation site (replication origin)
2. Expose single-stranded templates (unwind)
3. Load helicase at nascent fork
4. Prime DNA synthesis
Activities needed to initiate DNA replication
5’
origin
3’
5’
3’
helicase
DNA polymerase
Converting DS DNA to a replication fork
1. Recognize initiation site (replication origin)
2. Expose single-stranded templates (unwind)
3. Load helicase at nascent fork
4. Prime DNA synthesis
5. Load DNA polymerases and start DNA synthesis
Identifying origins genetically (replicator elements)
ARS Assay
Function: conferring autonomous maintenance on a plasmid
Using both Biochemistry and Genetics to understand function
Sequence
Element
recover
from cells
protein-DNA binding
introduce
into cells
Sequence
Element
in vitro
mutagenesis
Bacteria and Yeast have small well-defined origins
E. coli origin: 245 bp oriC
Initial
Unwinding
l
l
l
l
ORC Binding
Initiator DnaA
Loading
9
13 13 13
9
l l
l
9
l l l
S. cerevisiae origin: ~120 bp ARS1
9
9
A B1
Chromatin
Accessibility
B2
B3
l
9
DnaA 9-mer high affinity binding
13
A/T- rich 13-mer repeats
l
GATC sites (for regulation)
A and B1: ORC binding
B2 and B3: promote nucleosome free region?
Biochemical Dissection of OriC Initiation
Develop in vitro system
Establish “purified” system
DnaA
DnaB
DnaC
DnaG
Pol III holo
SSBP
Gyrase
Pol I, Rnase H, Ligase
Create partial reactions
- structurally analyze intermediates
- determine protein and cofactor dependencies
Infer protein function and develop specific assays
Simplified Model for oriC Initiation
DnaA
- binds origin, unwinds DNA, loads helicase
DnaB
- helicase
DnaC
- delivers and loads helicase
DnaG
- primes DNA synthesis
Pol III holo
- polymerase and clamp (polymerase) loading
SSBP
- stabilizes SS DNA
Gyrase
- negatively supercoil DNA (facilitates unwinding)
Pol I, Rnase H, Ligase - process Okazaki fragments
Bidirectional
Replication
ATPases Associated with a variety of cellular Activities (AAA+ proteins)
With ATP bound in subunit interfaces nucleotide state
can be coupled to large conformational change
Many replication proteins form
AAA+ ATPase Complexes
Arginine promotes
ATP hydrolysis
ATP Pocket
origin binding
DnaA
ORC
helicase loading
DnaC
Cdc6
helicase
DnaB
Mcm2-7
clamp loader
g complex RFC1-5
Schematic of Mcm2-7 hexamer organization
Oligomerization of DnaA-ATP bound to oriC promotes unwinding
high affinity binding
low affinity binding
oligomerization
SS DNA binding
Activities needed to initiate DNA replication
5’
origin
3’
5’
3’
helicase
DNA polymerase
Converting DS DNA to a replication fork
E. coli
1. Recognize initiation site (replication origin)
DnaA (oriC)
2. Expose single-stranded templates (unwind)
DnaA
3. Load helicase at nascent fork
DnaC, DnaA > DnaB
4. Prime DNA synthesis
Primase
5. Load DNA polymerases
Pol III Holoenzyme
Coordinating replication initiation with E. coli cell cycle
oriC
terminus
replication
initiation
Initiation occurs once per cell cycle at fixed ratio of cell volume per chromosome
- Wallden et al. 2016 Cell 166:729-739
Multiple mechanisms inhibit re-initiation of oriC
1) Origin Inactivation: Chromosomes are marked by dam methylation and
become temporarily hemimethylated when they are replicated. SeqA binding to
hemimethylated oriC blocks DnaA low affinity binding required for initiation
Me
GATC
CTAG
dam
GATC
CTAG
Me
dam
GATC
CTAG
Me
replication
SeqA binding to hemimethylated GATC prevents oriC re-initiation
oriC replication immediately leads to hemimethlyation of its GATC sites
SeqA binds specifically to hemimethlyated GATC, preventing DnaA-ATP binding to low
affinity binding sites in oriC and thereby blocking immediate re-initiation
SeqA binding also transiently protects hemimethylated GATC in oriC from dam methylase, but this
protection only lasts for 1/3 of the cell cycle, after which full methylation and SeqA release occurs
Coordinating replication initiation with E. coli cell cycle
oriC
terminus
replication
initiation
SeqA inhibition
by oriC inactivation
Multiple mechanisms inhibiting re-initiation of oriC
1) Origin Inactivation: Chromosomes are marked by dam methylation and
become temporarily hemimethylated when they are replicated. SeqA binding to
hemimethylated oriC blocks DnaA low affinity binding required for initiation
Me
GATC
CTAG
dam
GATC
CTAG
Me
dam
GATC
CTAG
Me
replication
2) Decreased Initiator Activity: DnaA-ATP is inactivated by ATP hydrolysis. It’s slow
intrinsic hydrolytic activity is greatly stimulated by
-- Hda1 bound to a loaded sliding clamp
-- binding to the datA sequence element in the presence of IHF protein
Regulation of DnaA activity via nucleotide binding
Binds oriC
Binds oriC
High Affinity Sites Low Affinity Sites
Unwinds
oriC
Load
helicase
DnaA-ATP
+
+
+
+
DnaA-ADP
+
-
-
-
DnaA
+
-
-
-
Appealing model: nucleotide driven molecular switch
DnaA-ATP is active initiator; DnaA-ADP and DnaA are inactive
DnaA-ATP hydrolysis and inactivation is coupled to initiation
DnaA-ADP release allows ATP rebind for next round of initiation
Hda: initiation induced activity that converts DnaA-ATP to DnaA-ADP
Initiation
Coordinating replication initiation with E. coli cell cycle
How is DnaA reactivated?
oriC
terminus
How is it coupled to
volume/chromosome ratio?
replication
initiation
Hda and datA
inhibition by DnaA
inactivation
SeqA inhibition
by oriC inactivation
Activities needed to initiate DNA replication
5’
origin
3’
5’
3’
helicase
DNA polymerase
Converting DS DNA to a replication fork
S. cerevisiae
1. Recognize initiation site (replication origin)
ORC
2. Expose single-stranded templates (unwind)
??? (Cdc45 - Mcm2-7 - GINS?)
3. Load helicase at nascent fork
ORC,Cdc6,Cdt1 > Mcm2-7
Sld2,Sld3,Dpb11 > Cdc45, GINS
4. Prime DNA synthesis
Pola-Primase
5. Load DNA polymerases
Sld2,Sld3,Dpb11 > Pole
???
> Pold
Bacteria and Yeast have small well-defined origins
E. coli origin: 245 bp oriC
Initial
Unwinding
l
l
l
l
ORC Binding
Initiator DnaA
Loading
9
13 13 13
9
l l
l
9
l l l
S. cerevisiae origin: ~120 bp ARS1
9
9
A B1
Chromatin
Accessibility
B2
B3
l
9
DnaA 9-mer binding
13
A/T- rich 13-mer repeats
l
GATC sites (for regulation)
A and B1: ORC binding
B2 and B3: promote nucleosome free region?
Identifying origins physically (mapping initiation sites)
1) Map the sites of earliest DNA synthesis in a region
2) Map sites where replication bubbles first form
Eukaryotic origins: lessons from yeast
- chromosomal context affects origin activity and timing
only some ARSs on plasmids are efficient origins in chromosomes
- multiple origins are spaced closely enough to back up each other
- but they are not redundant for long-term chromosome propagation
Multiple deletions do not grossly affect
chromosome replication and cell division
But increase the probability of rare rearrangements
ARSs
X
X
Origin Use
By 2-D Gel
X
1,000
kb
2,000
kb
S.cerevisiae Chromosome 3 Origins
3,000
kb
Metazoan origins may be defined by chromatin more than sequence
Sequence Recognition
In Yeast
In Metazoans
Chromatin Structure
ORC binds ACS
nucleosome free region
ORC binds nonspecifically
to AT rich sequences
chromatin accessibility may be
primary origin determinant
Better in vivo assays allow genetic analysis of eukaryotic replication
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)
In Vivo Assays for Protein DNA Interactions
Identifying intermediates in the assembly of initiation complexes on DNA
Chromatin IP (ChIP)
Preferred binding sites
of specific proteins
yORC1
ChIP preIP
control
control
Gel
ARS305
Microarray or
Sequencing
control
yORC1 ChIP-chip (chromosome VI)
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
Dynamic Protein Associations Through G1 and S
Combining temporal and spatial analysis of replication and binding in synchronized cells
Cdc45 ChIP-chip tracks with fork movement
Cell Cycle Time
BrdU incorporation monitors 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 d, DNA Pol e, 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 analysis of Mcm loading and activation
Pre-RC Assembly (helicase loading)
Mcm2-7 doublehexamer remains
on DNA after high salt wash
ORC-DNA
ATP
Helicase Activity
Drosophila extract
purify
helicase
activity
Cdc6
Cdt1-Mcm2-7
Cdc45 - Mcm2-7 - GINS
(CMG - helicase “holoenzyme”)
EM reconstruction
side
C
N
hexamer
N
end
C
hexamer
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
Cell Cycle Inputs: activation of CDKs and DDKs trigger origin initiation
Cdc7-Dbf4
Kinase
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 show a protein is a biologically relevant target of a 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 e, Dpb11
DDK
Mcm4 and Mcm6 are essential targets for Cdc7-Dbf4 kinase (DDK)
Regulation of eukaryotic replication initiation
 Dependence on completion of G1 phase
dependence on Cdc7 and CDK kinases
 Timing of origin firing within S phase
see slide in appendix
 Preventing re-initiation within a single cell cycle
example: origins/cell = 50,000
fidelity per origin = 0.9999999
fidelity per cell = (0.9999999)
50,000
= 0.995
Many mechanisms inhibit re-licensing and prevent re-initiation
origin activation
inhibit licensing
inhibit licensing
origin licensing
inhibit licensing
DDB1-Cul4
PCNA
GEMININ
Replication control is critical for genome stability
Gene Amplification
Aneuploidy
Other Instability?
Translocations?
Inversions?
Loss of Heterozgosity?
DNA replication is coordinated with other genomic processes
Replication of chromatin and chromatin states
-- nucleosome structure and chromatin states are disrupted by the replication fork
and must be faithfully duplicated after passage of the replication fork
-- changes in chromatin states are associated with changes in replication timing
Transcription
-- prokaryotic replisomes can survive collisions with the transcription machinery
-- but genomes still appear to avoid head on collisions between replication
and transcription from strongly expressed genes.
DNA Damage Repair
-- Replication provides genome surveillance
Sister Chromatid Cohesion
-- establishment of cohesion is coupled to DNA replication
Meiotic Recombination
-- the DS breaks that initiate meiotic recombination are coupled to DNA replication
Bioreg 2017 Lecture 3 Replication
APPENDIX
Yeast origins maintain a nucleosome free region
Nucleosome positions relative
to ORC binding sites aligned
for 219 origins
White – nucleosome occupied
Black – nucleosome free
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 selectable plasmid because of
higher chance for plasmid replication failure
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
Initiation or Elongation?: Execution Point Analysis by Reciprocal Shift
Requires independent and reversible means of inactivating two functions plus an “endpoint” assay
Initiation: mutated gene has executed its function by the time DNA synthesis occurs
DNA synthesis
1st shift ts
Experiment 1
2nd shift HU
1st shift HU
Experiment 2
2nd shift ts
Elongation: mutated gene is still needed to function during DNA synthesis
DNA synthesis
1st shift ts
Experiment 1
2nd shift HU
1st shift HU
Experiment 2
2nd shift ts
Origin initiation timing within S phase is regulated
Earlier Origins
Earlier
Replication
Later Origins
This timing correlate with transcriptional program and cell fate
Embryonic Stem Cells
Neural Precursor Cells
The CDK paradigm for once and only once replication
preRC assembly
NO preRC assembly
CDK
NO
triggering initiation
Sld2
Sld3
trigger initiation
The CDK paradigm for once and only once replication
preRC assembly
Some preRC re-assembly
CDK
NO
triggering initiation
Sld2
Sld3
trigger initiation
You have a 100 bp sequence element whose function you can quantitatively assay after introducing it into cells
You have a protein that you purified based on its ability to bind the sequence element and you wonder whether
this protein is required for the function of the sequence element.
Hence, you plan on identifying the gene that encodes the protein and mutagenizing the gene in cells where you
can functionally assay the sequence element
However, before you take all the effort to clone the gene and do the reverse genetics, you want some indication
that this binding protein is probably important for the function of the sequence element.
What quicker experiment can you do using available assays and reagents to increase your confidence that your
protein is functionally relevan