Lecture 3: Regulation through feedback inhibition by reaction products Analyzing role and function...
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Transcript of Lecture 3: Regulation through feedback inhibition by reaction products Analyzing role and function...
![Page 1: Lecture 3: Regulation through feedback inhibition by reaction products Analyzing role and function of sequence elements Origins and Initiation and Regulation.](https://reader033.fdocuments.us/reader033/viewer/2022052414/56649e415503460f94b33f3b/html5/thumbnails/1.jpg)
Lecture 3:
Regulation through feedback inhibition by reaction products
Analyzing role and function of sequence elements
Origins and Initiation and Regulation
Increasing the power of genetic tools with better in vivo molecular phenotypes
Regulation through cell cycle control inputs
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Prokaryotic and Eukaryotic Replication Initiation Activities
1. Recognize initiation site (replication origin)
2. Expose single-stranded templates (unwind)
3. Load helicase at nascent fork
4. Prime DNA synthesis
5. Load polymerase(s)
5 ’
5 ’
3 ’
3 ’
5 ’
3 ’5 ’
5 ’
3 ’
3 ’
Converting DS DNA to replication fork
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Identifying Replicators (Genetic Mapping of Origins)
ARS Assay
Function: conferring autonomous maintenance on a plasmid
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Bacteria have small well-defined origins
E. coli origin: 245 bp oriC S. cerevisiae origin: ~120 bp ARS1
13 13 13
A/T- rich 13-mer repeats
Initiator DnaALoading
9 9 9 9
DnaA 9-mer binding
InitialUnwinding
A B1 B2 B3
ORC Binding
A and B1: ORC binding
9
13
GATC sites (for regulation)
ChromatinAccessibility
B2 and B3: promote nucleosome free region?
9
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Create partial reactions and structurally analyze intermediates
Biochemical Dissection of OriC Initiation
dnaA - Initiator: bind origin, unwind DNA, load helicasednaB - helicasednaC - deliver and loads helicaseSSB - stabilizes unwound DNAdnaG - prime DNA synthesisgyrase - negatively supercoil DNA (facilitates unwinding)PolII holo - DNA synthesisPol I, Rnase H, Ligase -process Okazaki fragments
Develop in vitro system
Establish “purified” system
Infer protein function and develop specific assays
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Model for oriC Initiation
Bidirectional Replication
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ATP is an allosteric regulator of DnaA
oligomerizationSS DNA bindingunwinding
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Prokaryotic and Eukaryotic Replication Initiation Activities
1. Recognize initiation site (replication origin)
2. Expose single-stranded templates (unwind)
3. Load helicase at nascent fork
4. Prime DNA synthesis
5. Load polymerase(s)
5 ’
5 ’
3 ’
3 ’
5 ’
3 ’5 ’
5 ’
3 ’
3 ’
DnaA binds oriC
DnaA
DnaC loads DnaB
Primase
E. coliConverting DS DNA to replication fork
DnaB binds t subunitSSB & primer-template
bind Clamp-Loader & Clamp
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1) Origin Inactivation: Chromosomes are marked by dam methylation and become temporarily hemimethylated when they are replicated SeqA binding to hemimethylated oriC blocks DnaA initiation function
GATC dam GATC dam GATCCTAG CTAG CTAG
Me Me
Me
Multiple mechanisms inhibiting re-initiation of oriC
replication
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Exactly how seqA blocks oriC re-initiation is not known
seqA binding to hemimethlyated oriC somehow prevents dnaA initiation functionwithout inhibiting dnaA high affinity binding to oriC
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1) Origin Inactivation: Chromosomes are marked by dam methylation and become temporarily hemimethylated when they are replicated SeqA binding to hemimethylated oriC blocks DnaA oligomerization
GATC dam GATC dam GATCCTAG CTAG CTAG
Me Me
Me
2) Decreased Initiator Activity: DnaA-ATP is inactivated by ATP hydrolysis by -- Hda1 bound to a loaded sliding clamp -- binding to the DnaA binding element datA
Multiple mechanisms inhibiting re-initiation of oriC
replication
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Binds oriC
Regulation of DnaA activity via nucleotide binding
Unwinds oriC Load helicase
DnaA-ATP + ++
DnaA-ADP + - -
DnaA + - -
Appealing model: nucleotide driven molecular switch
DnaA-ATP is active initiator
DnaA-ATP hydrolysis and inactivation is coupled to initiation
DnaA-ATP is reset for next round of initiation
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Conversion of DnaA-ATP to DnaA-ADP
Levels of ATP bound to DnaA cycle: high (~80%) just before initiation and low (~16%) soon after
In vivo evidence for the conversion
Preventing ATP hydrolysis with a dnaA hydrolysis mutation or an hdaA deletion leads to
-- accumulation of DnaA-ATP
Genetic evidence for the relevance ATP hydrolysis
-- overreplication of DNA
HdaA stimulates DnaA ATPase activity when bound to a clamp loaded onto a primer-template junction
Biochemical purification of an ATPase stimulating activity
This couples inactivation of DnaA to a late initiation event
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The Challenge: coupling regulation of DnaA-ATP state to replication
initiation cycles
Clamp loading
RIDA Regulatory Inactivation of DnaA
DNA Synthesis
DDAH datA-dependent DnaA-ATP Hydrolysis
DARS DnaA Reactivating Sequence
IHF Integration Host Factor
HdaA
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Prokaryotic and Eukaryotic Replication Initiation Activities
1. Recognize initiation site (replication origin)
2. Expose single-stranded templates (unwind)
3. Load helicase at nascent fork
4. Prime DNA synthesis
5. Load polymerase(s)
5 ’
5 ’
3 ’
3 ’
5 ’
3 ’5 ’
5 ’
3 ’
3 ’
DnaA binds oriC ORC binds origins
DnaA
DnaC loads DnaB Cdc6 & Cdt1 load Mcm2-7
Primase DNA Pol - primase
E. coli S. cerevisiae
ORC? Mcm2-7?
Mcm10?Cdc45-Sld3Dpb11-Sld2GINS complex
Converting DS DNA to replication fork
DnaB binds subunitSSB & primer-template
bind Clamp-Loader & Clamp
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Budding yeast also have small well-defined origins
E. coli origin: 245 bp oriC S. cerevisiae origin: ~120 bp ARS1
13 13 13
A/T- rich 13-mer repeats
Initiator DnaALoading
9 9 9 9
DnaA 9-mer binding
InitialUnwinding
A B1 B2 B3
ORC Binding
A and B1: ORC binding
9
13
GATC sites (for regulation)
ChromatinAccessibility
B2 and B3: promote nucleosome free region?
9
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Yeast origins have a nucleosomal structure
Nucleosome positions relative to ORC binding sites aligned for 219 origins
White – nucleosome occupied
Black – nucleosome free
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Eukaryotic origins appear “redundant”
ARSs
Origin UseBy 2-D Gel
S.cerevisiae Chromosome 3 Origins
X X
X
Multiple deletions have little overall effecton chromosome replication and cell division
But increase the probability of rare rearrangements
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Identifying Sites of Initiation (Physical Mapping of Origins)
Example: Map the earliest DNA synthesis in a region
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Chromatin Structure
nucleosome free region
Sequence Recognition
ORC binds ACSIn Yeast
In MetazoansORC binds nonspecifically
to AT rich sequencechromatin may be primary origin determinant
Higher eukaryotic origins may be defined by chromatin
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Bioregulation through regulated protein assembly
Pre-RC Pre-ICPost-RC
Initiation
CDKCdc7-Dbf4
License Trigger
GINS
M Phase G1 Phase S Phase
2-stage model for eukaryotic replication initiation
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Develop in vitro system
Establish “purified” system
Create partial reactions and structurally analyze intermediates
Infer protein function and develop specific assays
Genetically identify initiation factors
A Tale of Two Systems
Localize factors to origins and/or replication forks
Develop in vitro system and specific assays
Establish order of assembly during initiation and cell cycle progression
Future mechanistic studies (great Bioreg proposals)
E. Coli oriC S. cerevisiae ARS
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Genetic Screens Enriching for Replication Initiation Mutants
Conditional Mutants:cell division cycle (cdc)
Hypomorphic Mutants:minichromosome maintenance (mcm)
budded morphology1N DNA content
faster loss of minichromosome (I.e. selectable plasmid) from population
suppression of mcm phenotype with multiple plasmid origins
cdc6mcm2mcm3mcm5/cdc46
execution point before elongation
cdc6cdc46/mcm5cdc47/mcm7cdc54/mcm4
cdc7dbf4cdc45
mcm10
% cells containing plasmidWITH selection
% cells containing plasmidwithOUT selection
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Initiation or Elongation?: Execution Point Analysis
2nd shift ts
A mutated initiation function is completed by the time elongation is blocked
Elongation
1st shift HU
Elongation
A mutated elongation function is still needed when elongation is blocked
1st shift HU
Requires independent and reversible means of inactivating two functions plus an “endpoint” assay
2nd shift ts
HU = hydroxyurea which blocks replication elongation by inhibiting dNTPs biosynthesis
Initiation
Initiation
Cell CycleCompleted
Cell CycleRemains Blocked
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A Yeast Initiator Protein: Guilt by Association
S. cerevisiae origin: ~120 bp ARS1
A B1 B2 B3
ORC Bindingon naked DNA
A is an essential ARS consensus sequence
Help ORCbind on chromatin
Biochem: Binding ActivityARS1 Footprint
Note: most other eukaryotic ORCs do NOT have such sequence specificity
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In Vivo Assays for Protein DNA InteractionsIdentifying intermediates in the assembly of initiation complexes on DNA
Chromatin IP (ChIP)Preferred binding sites
of specific proteins
Genomic FootprintProtein binding and/or
distortion of specific sites
AR
S1
DN
A
DN
A:y
OR
C
Gen
om
ic F
oo
tpri
nt
yORC1ChIP preIP
ARS305
control
control
control
Gel
yORC1 ChIP-chip (chromosome VI)
Microarray
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Pre-Replicative Complex (pre-RC) in G1 Phase Temporal analysis of genomic footprint at origins
M G1 S-G2-M
ORC hypersensitive sitereduced in G1 phase
Extended protectionof B domainIn G1 phase
Yea
st 2
µ o
rigin
Speculation: ORC binds origin throughout the cell cycle and is joined by other proteins in G1
phase to “license” origins for initiation
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Ordered Assembly of Proteins at Origins During G1 & S Using ChIP to establish temporal order and genetic dependencies of proteins assembling at the origin
ARS1
control
control
control
G1 S - G2 -MG1 S - G2 -M
- Cdc6 + Cdc6
preIP
Example: G1-specific recruitment of Mcm7 is dependent on Cdc6
time points sampled for Mcm7 ChIP
G1MG2SG1Synchronizedyeast culture
- Cdc6 or + Cdc6
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Dynamic Protein Associations Through G1 and S Combining temporal and spatial analysis of replication and binding in synchronized cells
Some replication proteins that load at origins later move with the forks:Mcm2-7, Cdc45, GINS, Mcm10, Dpb11, DNA Pol , DNA Pol , DNA Pol , PCNA
(clamp), RFC1-5 (clamp loaders), RFA
BrdU incorporation monitors fork movement Cdc45 ChIP-chip tracks with fork movement
Cel
l Cyc
le T
ime
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2-Stage Model for Protein Assembly During Replication Initiation
Pre-RC Pre-ICPost-RC
Initiation
License Trigger
GINS
M Phase G1 Phase S PhaseCDKCdc7-Dbf4
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Biochemical insights into Mcm loading and activation
Pre-RC Assembly Assay (helicase loading)
Can substitute mutant/modified proteins with altered activities
Can control addition order of protein, cofactors, or inhibitors
Can analyze structures with greater resolution and accuracy
ORC-DNA
Cdc6
Cdt1-Mcm2-7
ATP
Mcm2-7 doublehexamer remains on DNA after high salt wash
N
EM reconstruction
side end
C N C
hexamer hexamer
Helicase Activity
Drosophila extract
purifyhelicaseactivity
Cdc45 - Mcm2-7 - GINS(CMG - helicase “holoenzyme”)
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2-Stage Model for Protein Assembly During Replication Initiation
Pre-RC Pre-ICPost-RC
Initiation
License Trigger
GINS
M Phase G1 Phase S PhaseCDKCdc7-Dbf4
core helicase loaded around
DS DNA
helicase holoenzymeloaded around
unwound SS DNA
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Activation of CDKs and DDKs in S phase trigger origin initiation
Clb-Cdc28(CDK)
Dbf4-Cdc7(DDK)
Pre-RCPost-RC Pre-IC
Initiation
G2S
Cdc7-Dbf4Kinase
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Temporal control of DNA replication through earlier DDK action?
CDKDDK
Pre-RC (DDK activated)
Post-RC
Initiation
G2S
Post-RC
Pre-RC
DDK CDK
Pre-RC Initiation
EarlyOrigins
LateOrigins
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?
Cdc45Sld3
Cdc7-Dbf4Kinase
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Cell cycle control of origin function must be highly efficient
X X XX X X
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
(.9999999)50,000
= .995
CDK
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preRC assembly
NOtriggering initiation
NO preRC assembly
trigger initiation
CDK
Sld2 Sld3
The CDK paradigm for once and only once replication
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preRC assembly
NOtriggering initiation
Some preRC re-assembly
trigger initiation
CDK
Sld2 Sld3
The CDK paradigm for once and only once replication
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CDKs Target Multiple Proteins to Block pre-RC Re-assembly
In budding yeast, CDK phosphorylation of
1) Mcm3 promotes Mcm2-7 nuclear exclusion
2) Cdc6 promote its proteolysis
4) Orc2/Orc6 inhibits recruitment of Cdt1-Mcm2-7
Overlapping mechanisms ensure re-initiation is blocked at thousands of origins
3) Cdc6 promotes CDK binding and inhibition
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
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Gene Amplification
Partial loss of replication control in yeast can greatly induce genomic instability How important is it to prevent re-initiation?
Aneuploidy Other Instability?
Translocations?
Inversions?
Loss of Heterozgosity?