The initiation of yeast DNA replication

Questions?Contact michael.weinreich@vai.orgVan Andel Research InstituteGrand Rapids, MI 49503

BMB801 Lecture 16 -- Dr. Michael Weinreich 10/4/06

Initiation of DNA Replication

Essential for cell growth, development, integrity of the genetic information Each origin initiates replication only once per cell cycleHighly regulated. Commitment to DNA replication is an irreversible decisionMany initiation proteins are upregulated in cancer cells

Initiation of DNA Replication

QUESTIONS

1. What are the requirements for the initiation of DNA replication?

2. How is initiation limited to once per cell cycle?

3. What effect might chromatin structure have on initiation?

4. Overview of the temporal nature of replication initiation

Essential for cell growth, development, integrity of the genetic information Each origin initiates replication only once per cell cycleHighly regulated. Commitment to DNA replication is an irreversible decisionMany initiation proteins are upregulated in cancer cells

origin

DNA Replication Begins at Specific Sequences

Fiber AutoradiographyVisualize 3H-Thymidine incorporation following a short pulse

origin

Isolation of Autonomously Replicating Sequences

Restriction digest of yeast chromosomal DNA

“Shotgun” clone into a URA3 origin-less plasmid

Select for URA+ cells

Transform into ura3 yeast

Isolation of Autonomously Replicating Sequences

EcoRI

EcoRI

URA3URA3URA3

URA+

Isolation of Autonomously Replicating Sequences

EcoRI

EcoRI

URA3URA3URA3

Isolation of Autonomously Replicating Sequences

EcoRI

EcoRI

URA3URA3URA3

Ura-

Isolation of Autonomously Replicating Sequences

EcoRI

EcoRI

URA3URA3URA3

-URA plate

URA+

ARS elements consist of two domains

A { BARS element~150-200bp

ARS elements share a common sequence - the ACS

A { BARS element~150-200bp

WTTTAYRTTTW

W= A or T

R= A or G

Y= T or C

ARS Core Consensus Sequence

Plasmid stability measurements of linker scan mutants

A { BARS element~150-200bp

1. Transform plasmid into yeast selecting for URA3 marker Failure to recover high frequency of transformation (HFT) - essential sequences2. Grow yeast in medium lacking uracil to get a population of cells3. Grow in medium containing uracil to allow plasmid loss events4. Calculate percentage of cells containing plasmid after X generations

“GGTCGAC”SalI

0

10

20

30

40

50

60

70

80

90

LS1 LS2 LS3 LS4 LS5 LS6 LS7 LS8 LS9LS10LS11LS12LS13LS14LS15LS16LS17LS18LS19LS20LS21LS22LS23 LS24LS25LS26LS27LS28LS29LS30LS31LS31.5

LS32LS33LS34LS35LS36

A B1 B2 IS

Linker scan analysis of ARS315

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +_ _+ + HFT

ACS

Percentage of plasmid containing cells

ARS1A B1 B2 B3

Linker scan analysis reveals modular structure of origins

ACS

ARS307

Essential Important

ARS1

ARS315

ARS305

A B1 B2 B3

There are at least two broad classes of origins in S. cerevisiae

ACS

ISA B1 B2

ARS307

?Inhibitory element

Jacob and Brenner’s Replicon Model

WTTTAYRTTTW

Initiator protein

Replicator

Jacob, F., and S. Brenner. 1963. [On the regulation ofDNA synthesis in bacteria: the hypothesis of the replicon.]C R Hebd Seances Acad Sci 256: 298-300.

Jacob and Brenner’s Replicon Model

Initiator protein -- DnaA

Replicator -- OriC13mers

DnaA boxes

E. coli

Jacob and Brenner’s Replicon Model

Initiator protein -- ORC(Origin Recognition Complex)

Replicator -- ARS

Bell, SP and Stillman, B. (1992) ATP-dependent recognition of eukaryoticorigins of DNA replication by a multiprotein complex.Nature 357(6374):128-34.

A B1 B2 B3

ORC binds to origins of replication

origin

A B1 B2

ORC

-ORC is a six-subunit protein complexthat binds to ARS elements.

-Specific DNA binding requires ATP

-All subunits are essential and are required for initiation

-ORC binds ARS elements throughout the cell cycle

…and then recruits Cdc6p and Cdt1p during early G1

origin

A B1 B2

ORC

A B1 B2

ORC

Cdc6p

Cdt1p

II III IV V VISensI SensII

VII

Walker A Walker BLSGAPGTGKTAC..{}..IVLVLDEMDQL HumanISGAPGTGKTAC..{}..ILLVLDEMDQL XenopusVSGAPGTGKTVL..{}..VIIVLDEMDHL S. pombeITGPPGTGKTAQ..{}..FVVVLDEMDRL S. cerevisiaeis GaPGTGKTac..{}..ivl VLDEMDqL Consensus

AAR, A or E

Cdc6 is a AAA+ protein required for pre-RC assembly

K114A mutant is ts: cdc6-4

MCM

A B1 B2

ORC

pre-RC

The “pre-Replicative Complex” assembles at all origins in G1

MCM

A B1 B2

ORC

A B1 B2

ORC

Cdc45p

Cdc7p-Dbf4ppre-RC

Kinases then activate the initiation of DNA synthesis

Cdk1-Clb+

GINS

Ser/Thr protein kinases

MCM

A B1 B2

ORC

Cdc45p

Origin unwinding occurs

GINS

MCM

A B1 B2

ORC

… followed by the assembly of DNA polymerases

Cdc45p

Pol primase

GINS

and bi-directional DNA synthesis

Cdc6

ORC

Pre-RCAssembly

MCMs

Cdc7 Kinase

G1 S-PhaseM

ORC

ORC

Initiation

P

P

P

P

ORC

OriginMarking

Initiation of DNA Replication

DNA Polymerase

Cdk KinaseCdc7 Kinase

Cdc45Cdt1

P

P

Cdk Kinase

How do cyclin-dependent kinases prevent pre-RC formation?

CyclinB-Cdks phosphorylate ORC, Cdc6p and MCM proteins

ORC phosphorylation inhibits its activity through an unknown mechanism

Cdc6p phosphorylation causes is proteolysis

MCM phosphorylation excludes it from the nucleus, as well as Cdt1p

MCM

A B1 B2

ORC

pre-RCLow Cdk levels

A B1 B2

ORC

PP

P

High Cdk levels

Cdc6

ORC

Pre-RCAssembly

MCMs

Cdc7 Kinase

G1 S-PhaseM

ORC

ORC

Initiation

P

P

P

P

ORC

OriginMarking

Initiation of DNA Replication

DNA Polymerase

Cdk KinaseCdc7 Kinase

Cdc45Cdt1

P

P

Cdk Kinase

ORC and Abf1p position nucleosomes outside ARS1

A B1 B2 B3

ORCAbf1p

… facilitating pre-RC formation at the origin

A B1 B2 B3

ORCAbf1p

Cdc6p

MCMCdt1p

Origins lacking a B3 element may be sensitive to nucleosome intrusion

A B1 B2 B3

ORCAbf1p

A B1 B2

ORC

Cdc6p

MCMCdt1p

A B1 B2 B3

ORCAbf1p

A B1 B2

ORC

…that inhibits pre-RC assembly

Cdc6p

MCMCdt1p

Cdc6pCdt1p

ARS1

ARS315

ARS305

A B1 B2 B3

ACS

ISA B1 B2

ARS307

?

nucleosome

IS element may position a nucleosome over origin

300301

302303320

304 305 306 307308

309 310 313 315 316 317 319314 318

Chromosome III

Position of ARS elements along chromosome III

< 10% < 10%Inactive

300301

302303320

304 305 306 307308

309 310 313 315 316 317 319314 318

Chromosome III

Position of ARS elements along chromosome III

< 10% < 10%Inactive

Early origins

300301

302303320

304 305 306 307308

309 310 313 315 316 317 319314 318

Chromosome III

Position of ARS elements along chromosome III

< 10% < 10%Inactive

Late replicatingTelomeres

Early origins

Replication timing along chromosome VI

Raghuraman MK et al. (2001) Replication dynamics of the yeast genomeScience 294(5540):115-21.

DNA damage during S-phase, telomeres, late replication and the centromere

Questions?Contact michael.weinreich@vai.orgVan Andel Research InstituteGrand Rapids, MI 49503

BMB801 Lecture 17 -- Dr. Michael Weinreich 10/6/06

ARS1A B1 B2 B3

Linker scan analysis reveals modular structure of origins

ACS

ARS307

Essential Important

Plasmid stability measurements of linker scan mutants

A { BARS element~150-200bp

“Linker scans” are typically ~6-10bp in length. They change the existing sequenceat multiple base pairs without the adding or deleting nucleotides and introduce anovel restriction site. For example, an 8bp XhoI linker scan across this region mightchange the existing sequence to CCTCGAGG at each position above.

Formation of the pre-RC occurs during G1

origin

A B1 B2

ORC

A B1 B2

ORCMCM

A B1 B2

ORC

Cdc6p

Cdt1p

MCM, DNA helicaseORC, Origin Recognition Complex

Cdc6p and Cdt1p, helicase loaders

MCM

A B1 B2

ORC

A B1 B2

ORC

Cdc45p

Cdc7p-Dbf4ppre-RC

Kinases then activate the initiation of DNA synthesis

Cdk1-Clb+

GINS

Ser/Thr protein kinases

MCM

A B1 B2

ORC

… by promoting the assembly of DNA polymerases

Cdc45p

Pol primase

GINS

and bi-directional DNA synthesis

QUESTIONS

5. What determines the temporal order of replication during S-phase?

6. How might DNA damage affect DNA replication?

7. What are telomeres and how are they replicated?

8. Overview of the centromere and kinetochore

Late replication, the telomere and functions of the centromere

MCM

A B1 B2

ORC

pre-RC

The “pre-Replicative Complex” assembles at all origins in G1

Early and late origins assemblethe pre-RC during G1

Since some origins are not activateduntil late S-phase, the regulatory stepmust occur after pre-RC formation

Cdc7-Dbf4 and Cdk1-Clb5,6?

Replication timing along chromosome VI

Raghuraman MK et al. (2001) Replication dynamics of the yeast genomeScience 294(5540):115-21.

MMS activates the intra-S-phase checkpoint which inhibits late origin firing

Mec1p (ATR)

Rad53p (Chk2)Chk1p

Mec2/Ddc1(ATRIP)

Rad9p

DNA damage (MMS)

G2/M Inhibit lateorigin firing

WT

origin

MMS activates the intra-S-phase checkpoint which inhibits late origin firing

Mec1p (ATR)

Rad53p (Chk2)Chk1p

Mec2/Ddc1(ATRIP)

Rad9p

DNA damage (MMS)

G2/M Inhibit lateorigin firing

WT

+MMS

origin

Slows S-phase

Cdc7p-Dbf4p

Dbf4p is phosphorylated following replication arrest

MCM

A B1 B2

ORC

Cdc45p

Pol primase

GINS

DNA damageReplication arrest

Mec1p

Rad53pLate origins

Dbf4p-Cdc7p

P

?pre-RC

Cdc7p-Dbf4p

Homologous proteins in S. pombe, Xenopus, mouse and humans

Cdc7p is a serine/threonine kinase required for entry into S phase… after assembly of the pre-RC

Dbf4p is a regulatory subunit required for kinase activity

Phosphorylates MCM proteins, Cdc45p and polymerase- primase in vitro

Cdc7p-Dbf4p is required for loading Cdc45p and GINS at the origin

MCM

A B1 B2

ORC

A B1 B2

ORC

Cdc45p

Cdc7p-Dbf4ppre-RC

Cdk1-Clb+

GINS

1 704350

N M C

DBF4 contains three regions of homology among diverse species

Is the N-terminus required for viability (DNA replication) or DNA repair?

What role might Dbf4p play in checkpoint pathways for DNA repair?

Dbf4p

The DBF4 N-terminal conserved domain is not required for viability

N M C

Δ65 Δ87Δ109

Δ128Δ136

Δ158Δ172

Δ186Δ206

Δ229Δ265

Δ136-221Δ71-221

viable non-viable

Dbf4p

NLSΔ292

Δ221

NLS

NLS

NLS NLS

135 179 260 309 656 697BRDF

Δ188-221

BRDF= BRCT and DBF4 similarity

Loss of the BRCT-like domain causes defect in response to DNA damage

1 704350

BRCT M C

1. Promotes DNA repair2. Is required for firing late replication origins

Cdc7p-Dbf4p

Dbf4p

Essential domain

BRCT domains are present in proteins involved in the DNA damage responseProtein interaction domains - phospho-peptide binding

Mec1p and Rad53p phosphorylate Dbf4p following exposure to HU

Mec1p (ATR)

Rad53p (Chk2)Chk1p

Dun1p

Tel1p (ATM)

Ddc2(ATRIP)

Cdc5p(Plk)

Dbf4p

Bub2pAnaphase Crt1

Securin

Tem1p(GTPase) Mitotic Exit

WT

rad53-1

0 1 2hrs in HU

Dbf4p

Cdc7p

DNA repair genes

Late origins?

DNA damage

Slows DNA replication

Telomeres occur at the ends of chromosomes

3’5’

Nucleosomal Non-nucleosomal

TG(1-3) budding yeast

T2AG3 human cells

Repeated DNA sequence

}

G-rich

The “End Problem”

DNA polymerases require a template primer to synthesis DNANo de novo DNA synthesis

Polymerases only synthesize DNA in the 5’ to 3’ direction

DNA primase can synthesize a short RNA primer without a template, but RNAsare removed during DNA synthesis because they are unstable

5’ 3’

3’

5’

RNA

Leading

Lagging

The “End Problem”

DNA polymerases require a template primer to synthesis DNANo de novo DNA synthesis

Polymerases only synthesize DNA in the 5’ to 3’ direction

DNA primase can synthesize a short RNA primer without a template, but RNAsare removed during DNA synthesis because they are unstable

5’ 3’

3’

5’

RNA is removedleaving a gap

5’ 3’

If not repaired, chromosomes would shorten over time

“Telomere erosion”

5’ 3’

Telomerase is a specialized polymerase that maintains telomere length

5’ 3’

Telomerase is a specialized polymerase that maintains telomere length

Telomerase contains an RNAthat serves as a template forDNA synthesis

Telomerase is an RNA-directed DNA polymerase

“Reverse Transcriptase”

Telomerase consists of an RNA and a protein component

TLC1 is a 1.2kb RNA

Est2p is the catalytic subunit of telomeraseand belongs to the reverse transcriptase family

Telomerase in a Ribonucleoprotein “RNP”

Telomerase synthesis at the ends of chromosomes

Vega et al. (2003) Nat. Rev. MCB 4:948-959

Telomere associated proteins in human cells and budding yeast

Blackburn (2001) Cell 106:661-673

Telomere loops and folding

Vega et al. (2003) Nat. Rev. MCB 4:948-959

Conservation of telomerase and associated proteins

Vega et al. (2003) Nat. Rev. MCB 4:948-959

Telomere associated proteins in human cells and budding yeast

Rap1p is a DNA binding proteinthat recognizes telomeric DNA

Rap1p recruits the SIR complex

SIR complex recruits Ku

Blackburn (2001) Cell 106:661-673

The ssDNA-binding protein Cdc13p recruits telomerase to the ends

Cdc13p binds ssDNA and also interacts with Est1p

Est1p is a subunit of telomerase that binds to Est2

Can bypass requirement for Est1pby making a Cdc13p-Est1p fusion

Cdc13pTelomerase

Est1p

Est2p interaction

Blackburn (2001) Cell 106:661-673

Ku tethers telomeres to the nuclear periphery in budding yeast

In yeast and more complex eukaryotes, telomeres are clustered togetherand interact with the nuclear periphery

In the absence of Ku, telomeres are dispersed throughout the nucleusand no longer show this clustering arrangement

Sir2-4 collaborate to form heterochromatin adjacent to telomeres

Rap1Rap1

Sir3-4

Telomere

Rap1

Sir4p

Nucleosomal DNA

Sir4p also recruits the Sir2p histone deacetylase

Rap1p binds to telomeric DNA and recruits Sir3p-Sir4p

Sir2-4 collaborate to form heterochromatin adjacent to telomeres

Rap1Rap1

Sir3-4

Telomere

Rap1

Sir4p

Nucleosomal DNA

Sir2p

Sir4p also recruits the Sir2p histone deacetylase

Rap1p binds to telomeric DNA and recruits Sir3p-Sir4p

Sir2-4 collaborate to form heterochromatin adjacent to telomeres

Rap1Rap1

Sir3-4

Telomere

Rap1

Sir4p

Nucleosomal DNA

Sir2p

Sir4p also recruits the Sir2p histone deacetylase

Rap1p binds to telomeric DNA and recruits Sir3p-Sir4p

Sir3p-Sir4p bind to ‘hypoacetylated” histones H3 and H4

The Sir2p deacetylase is conserved

Yeast Bacterial Human

Sir2-4 collaborate to form heterochromatin adjacent to telomeres

Rap1Rap1

Sir2-4Sir2-4

Telomere

Rap1

Sir4p

Sir2-Sir3-Sir4 binding leads to deacetylation of an adjacent nucleosome

Sir2-4 collaborate to form heterochromatin adjacent to telomeres

Rap1Rap1

Sir2-4Sir2-4Sir2-4Sir2-4

Telomere

Rap1

Sir4p

Sir2-Sir3-Sir4 binding leads to deacetylation of an adjacent nucleosome

…. and results in the spreading of the SIR complex along chromatin

Sir2-4Sir2-4

Sir2-4 collaborate to form heterochromatin adjacent to telomeres

Rap1Rap1

Sir2-4Sir2-4Sir2-4Sir2-4

Telomere

Rap1

Sir4p

Sir2-Sir3-Sir4 binding leads to deacetylation of an adjacent nucleosome

…. and results in the spreading of the SIR complex along chromatin

Sir2-4Sir2-4

Some ARS elements are within the boundaries of this heterochromatin

Rap1Rap1

Sir2-4Sir2-4Sir2-4Sir2-4

Sir2-4Sir2-4

Telomere

Rap1

Sir4p

ARS

300301

302303320

304 305 306 307308

309 310 313 315 316 317 319314 318

Chromosome III

Position of ARS elements along chromosome III

< 10% < 10%Inactive

Late replicatingTelomeres

Early origins

Sir2-4Sir2-4

Rap1Rap1

Sir2-4Sir2-4Sir2-4Sir2-4

Sir2-4Sir2-4

Telomere

Rap1

Sir4p

ARS

Loss of SIR complex advances replication timing at telomeres

Ku

Rap1Rap1

Telomere

Rap1

Sir4p

ARS

(late)

(early)

sir3∆

WT

Ku

Sir2-4Sir2-4

Rap1Rap1

Sir2-4Sir2-4Sir2-4Sir2-4

Sir2-4Sir2-4

Telomere

Rap1

Sir4p

ARS

Ku

(late)

Ku70∆ advances replication timing at sub- & telomeric ARSs

WT

ARS501 (late)

Sir2-4Sir2-4

Rap1Rap1

Sir2-4Sir2-4Sir2-4Sir2-4

Sir2-4Sir2-4

Telomere

Rap1

Sir4p

ARS

Ku

Rap1Rap1

Telomere

Rap1

Sir4p

ARS

(late)

ku70∆

(early)

Ku70∆ advances replication timing at sub- & telomeric ARSs

Rif1p

ARS501 (early)

WT

ARS501 (late)

The centromere and kinetochore

CdeI CdeII CdeIII125bp

Centromere

The centromere and kinetochore

CdeI CdeII CdeIII125bp

Centromere

The “Kinetochore” is a proteinaceousstructure built upon the centromerefor microtubule capture and chromosomesegregation

~60 polypeptides

The relatively simple centromere of S.cerevisiae is located on an essential 125-bpregion of DNA comprising three functionalDNA elements. cdeII is AT-rich and can bind tothe centromeric nucleosome Cse4/CENP-A; however, the primary determinant of centromerelocation is cdeIII, which is bound by CBF3 and essential for the localization of all other kinetochoreproteins. cdeI is bound by CBF1 and not essential,but its deletion results in chromosome loss. In S.cerevisiae, homologues of three human foundationkinetochore proteins, Mif2/CENP-C, Mtw1/MIS12and Nnf/CENP-H, exist as part of larger multi-protein complexes – an arrangement that mightalso apply to higher eukaryotes.

Linear models of centromere organization

Trends Cell Biol. 2004 Jul;14(7):359-68.Amor DJ, Kalitsis P, Sumer H, Choo KH

Three dimensional view of a human centromere/kinetochore

Trends Cell Biol. 2004 Jul;14(7):359-68.Amor DJ, Kalitsis P, Sumer H, Choo KH

The spindle checkpoint prevents mitosis (anaphase) until all kinetochores attach

Mad2p

Mad2p-Cdc20p

(Inactive)

Cdc20p is a key mitoticregulator that is keptinactive until all kinetochoreshave attached to microtubules

MAD1, 2, 3BUB1, 3MPS1

Spindle Checkpoint genes

Spindle checkpoint prevents anaphase onset by inhibiting cohesin degradation

Musacchio and HardwickNat Rev MCB (2002)