Lecture 3: Regulation through feedback inhibition by reaction products Analyzing role and function of sequence elements Origins and Initiation and Regulation.

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

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

Identifying Replicators (Genetic Mapping of Origins)

ARS Assay

Function: conferring autonomous maintenance on a plasmid

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

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

Model for oriC Initiation

Bidirectional Replication

ATP is an allosteric regulator of DnaA

oligomerizationSS DNA bindingunwinding

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

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

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

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

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

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

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

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

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

Yeast origins have a nucleosomal structure

Nucleosome positions relative to ORC binding sites aligned for 219 origins

White – nucleosome occupied

Black – nucleosome free

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

Identifying Sites of Initiation (Physical Mapping of Origins)

Example: Map the earliest DNA synthesis in a region

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

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

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

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

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

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

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

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

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

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

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

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”)

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

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

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

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

preRC assembly

NOtriggering initiation

NO preRC assembly

trigger initiation

CDK

Sld2 Sld3

The CDK paradigm for once and only once replication

preRC assembly

NOtriggering initiation

Some preRC re-assembly

trigger initiation

CDK

Sld2 Sld3

The CDK paradigm for once and only once replication

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

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?