Lecture 3:

Regulation through feedback inhibition by product

Analyzing role and function of sequence elements (sequence specific assays)

Origins and Replication Initiation

5’

5’

3’

3’

5’

3’5’

5’

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

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

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?

Converting DS DNA to replication fork

DnaB binds subunitSSB & primer-template

bind Clamp-Loader & Clamp

Sld2, Sld3, Dpb11 loadCdc45 & GINS

Sld2, Sld3, Dpb11 loadsDNA Pol complex

?? loadsDNA Pol complex

2-Stage Model for Protein Assembly During Replication Initiation

Pre-RC Pre-ICPost-RC

Initiation

CDKCdc7-Dbf4

License Trigger

GINS

M Phase G1 Phase S Phase

Genetic Screens Enriching for Replication Initiation Mutants

Conditional Mutants:cell division cycle (cdc)

budded morphology1N DNA content

execution point before elongation

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 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

Genetic Screens Enriching for Replication Initiation Mutants

Conditional Mutants:cell division cycle (cdc)

budded morphology1N DNA content

execution point before elongation

cdc6cdc46/mcm5cdc47/mcm7cdc54/mcm4

cdc7dbf4cdc45

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

% cells containing plasmidWITH selection

% cells containing plasmidwithOUT selection

cdc6cdc46/mcm5cdc47/mcm7cdc54/mcm4

cdc7dbf4cdc45

mcm10

genetic & physical interactions with replication genes/proteins

POL2

DPB2DPB11

high copysuppression

PSF1coIP & mass spec PSF2

PSF3GINS complex

Gathering More Suspects: Guilt by Association

SLD4 = CDC45

SLD2

SLD3

SLD5

syntheticlethality

SLD1 = DPB3

DNA Pol subunits

Example: How Araki found many of the genes required for triggering initiation

CMG helicase holoenzyme subunits

Loaders for Pol Cdc45 & GINS

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 ActivityARS1 Footprint

Note: most other eukaryotic ORCs do NOT have such sequence specificity

Genetics: Establishing Initiation Function2-D gel analysis of ARS1 initiation

ORC5 orc5-1

Genetics: Hint from Mutational Correlation

ARS mutations

Poor in vitro binding activity

Poor in vivo origin function

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

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

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

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 (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”)

Replication Elongation Blocking

Discussion Paper

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

A Simplified View of Cyclin Dependent Kinases (CDKs)

CDK regulation

Molecular Biology of the Cell, 4th Ed.

different cyclins activate CDKs to promotedifferent cell cycle events

CDKs = kinase + cyclin

cyclins undergo periodic synthesis and proteolysis

CDK in S. cerevisiae

kinase = Cdc28

G1 cyclins = Cln1-3

G1 cyclinsnot shown

S & M cyclins = Clb1-6

Activation of CDKs and DDKs in S phase trigger origin initiation

Clb-Cdc28(CDK)

Dbf4-Cdc7(DDK)

Pre-RCPost-RC Pre-IC

Initiation

G2S

Identifying CDK and DDK targets for replication initiation

How to identify in vivo targets of kinase?

1) Kinase substrate in vivo

2) (Necessity) Phosphorylated sites essential for kinase function

phosphorylated in vivo in kinase dependent manner

in vitro and in vivo phosphorylation sites overlap

kinase substrate in vitro

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

Mcm4 and Mcm6 are essential targets for Cdc7-Dbf4 kinase (DDK)

DDK

A temporal program regulates DNA replication within S phaseLocate earliest DNA synthesis

One example: Microarray analysis of copy number

Earlier Initiation

Later Initiation

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

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

MCM2-7

Cdt1ORC

Cdc6

ORC

ORC

Fork Fork

ORC

ORC

ORC

assemblepre-RC

triggerinitiation

G2 M G1 S G2 M G1Start

preRC assembly

NOtriggering initiation

NO preRC assembly

trigger initiation

CDK

MCM2-7Cdc6ORC Cdt1

Sld2 Sld3

The CDK paradigm for once and only once replication

G2 M G1 S G2 M G1Start

preRC assembly

NOtriggering initiation

Some preRC re-assembly

trigger initiation

CDK

MCM2-7

ORCCdc6 Cdt1

ORC

ORC

Fork Fork

ORC

ORC

assemblepre-RC

triggerinitiation

ORC

ORC

Fork Fork

MCM2-7

ORCCdc6 Cdt1

MCM2-7Cdc6ORC Cdt1

X X X X

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

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

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?

QuickTime™ and a decompressor

are needed to see this picture.

Segurado & Tercero, Biol. Cell (2009) 11:617-627

DNA lesions induce responses to: (1) protect stalled forks (2) bypass lesions (3) delay further initiation (4) block cell cycle

Beyond Initiation: Keeping the Fork Going Through Thick and ThinMany genomic insults are now thought to originate from replication accidents

1

2

3

4

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?

Sister Chromatid Cohesion

-- how is the establishment of cohesion coupled to DNA replication?

Development and Differentiation

-- do replication timing changes help execute developmental decisions?

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 ProceedIf Replication Is Delayed or Blocked

Start

mitosis

Replication

elongation

initi

atio

n

Segregation

termination

Mec1

Rad53

Passage through Start independently sets into motion both Replication and Segregation

mec1

rad53

Branched view of cell cycleStandard view of cell cycle

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 ForksAbnormal replication intermediates accumulate when replication is delayed in checkpoint mutants

WT

CheckpointMutant(rad 53)

Unperturbed Replication dNTP Depleted Replication

hemi-replicated large gaps collapsed forks

Possible functions of checkpoint mechanisms at forks during replicative “stress”

maintain proper coordination of leading and lagging strand synthesisprevent fork collapse and branch migration

Based on EM analysis by Sogo & Foiani Labs, Science (2002) 297:599

These protective functions allow forks to resume replicating when the stress is removed

Origin Usage is Regulated During Development

Developing Frog S Phase Length

20-25 min

~ 6-8 hrs

~ 10 kb interorigin distance

~ 100 -200 kb interorigin distance

~ 20 kb interorigin distance

Line represents DS DNA

Origin Timing is Influenced by Chromatin Structure

ARS501late

ARS1early

Chr 5

Chr 4

ARS501

lateARS1

early

Chr 5

Chr 4

Position effect on origin timing

Swap positions

Telomeric heterochromatin can delay origin firing

from Brewer and Fangman labs, Cell (1992) 68:333

S. cerevisiae origins fire throughout S phase in a defined and reproducible order

TEL

Sir dependent heterochromatin

Y’ ARS fires late

TEL

Y’ ARS fires early

WT

sir mutant

from Gottschling Lab, Gene & Dev (1999) 13:146

Replication is Coupled to Establishment of Sister Chromatid Cohesion

Cohesin complexes (Scc1,Scc3,Smc1,Smc3) hold sisters together and ensure bipolar spindle attachment

Replication-like proteins have been implicated in the establishment of cohesion:

Cohesins must be present on chromatin during replication for proper cohesion to occur

Speculative polymerase switch model

- two DNA polymerases of the family,Trf4 and Trf5

- a modified RFC clamp loader with Ctf8,Ctf18, and Dcc1 substituting for Rfc1

figure from Hieter Lab, Mol Cell (2001) 7:959

Duplication of Nucleosome Structure During Eukaryotic Replication

Parental nucleosomes distribute randomly to daughter DNA

New histones complete the daughter complement of nucleosomes

Histone Deposition Factors (CAF-1 and/or Asf1)are recruited tonewly replicated DNA by PCNA and deposit H3H4 tetramers

How higher order chromatin structure is duplicated is not known

H2A-H2B dimers self assemble onto H3-H4 tetramers

Model for new histone deposition at replication forks

caf-1 asf1 double deletion mutants are viable in yeast,suggesting other histone deposition mechanisms exist

Does the replicator model apply to metazoan origin?

The ARS plasmid assay has failed to identify metazoan origins

Alternative strategy:

100 kb

AGE

example: potential origin in human globin gene cluster

LCR

LCR = Locus Control Region (required for transcriptional activity of entire cluster)

IR

IR = Initiation Region based on physical mapping of nascent strands

Deletions in either region(from thallasemia patients) inactivates the IR

physically map sites of initiationgenetically identify sequences required for initiation at those sites

Alternative hypothesis:

little sequence specificity for initiation sitezones of initiation established by chromatin structure

Eukaryotic Replication Initiation is Coupled to the Cell Cycle

Two fundamental stages of initiation defined primarily by Chromatin IP and Chromatin Association assays

pre-Replicative Complex(pre-RC)

Replicative Complex(RC)

1 2

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

Initiator

Helicase(?)

Origin RecognitionComplex

Helicase(?)Loaders

Kinases

Primase

Polymerase

Using both Biochemistry and Genetics to understand function

Nucleic Acids

Structural Analysis of Intermediates

Complexes

Size

Shape

DS versus SS

Topology

Modification

Covalent Linkages

Proteins

Modification

Ligand Binding

Conformation

Covalent Linkages

Composition

Stoichiometry

Conformation

Interacting SequencesStrand Pairing

Examples of structural features of intermediates that can be monitored

Cofactor (NTP) Status

Interacting Domains

2-Stage Model for Protein Assembly During Replication Initiation

Pre-RC Pre-ICPost-RC

Initiation

CDK Cdc7-Dbf4

License Trigger

GINS

M Phase G1 Phase S Phase

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

Fidelity: > 99.999% block for genome with 50,000 origins

X X X

Hayles et al. Cell 1994 Cyclin dependent kinases implicated