COS cells (infected with SV40)(expresses T Ag)

+ plasmid + dNTP + rNTP+ 32PdATP

37o C, 60 min

Analyze products by electrophoresis and autoradiography

SV40 ori

SV40 - + ori - +

Li & KellySV40 DNA replicationIn vitro.PNAS (1984) 81:6973

What else could they doto validate their system?

Fractionation to homogeneity

COS cells (infected with SV40)(expresses T Ag)

T Antigen frombaculovirus expression system

Human HeLa or 293 cells

cell lysate

PC

0.2 M KCl 0.66 M KCl

QS

0.2 M KCl 0.4 M KCl (PCNA)

ssDNA

0.6 M KCl 1 M KClRPA

+

(RF-C, pol + pol )

Prokaryotic Function Eukaryotic

SSB ss DNA coating RPA

PRIMASE RNA primer synth. Primase subunitof DNA POL ,

5'-3' polymerase DNA POL ,DNA POL

POL III CORE 3'-5' exonuclease DNA POL 5'-3' exonuclease Fen1

COMPLEX ATP dependentclamploader

RF-C

CLAMP processivity factor PCNA

LIGASE seal nicks Lig1

DnaB Helicase ?

ELONGATION FACTORS ARE CONSERVED

Subunit

Gene Bacterial Function Eukaryotic

dnaE | 5'-3'polymerase

DNA POL

dnaQ(mutD)

| POL IIICORE

3'-5'exonuclease

DNA POL

| 5'-3'exonuclease

Fen1

dnaX

dnaX | | ATP' |

COMPLEXdependentclamploader

RF-C

| |

dnaN CLAMP processivityfactor

PCNA

CONSERVATION FROM PROKARYOTES TOEUKARYOTES

RF-C is a five-subunit complexAll subunits are related in sequence and have ATP binding motifsATP hydrolysis by RF-C is associated with the loading of PCNARF-C is the functional homolog of the clamp-loader complex

RF-C

RF-C

Polymerase switching occurs even on lagging strands; pol does most of DNA synthesis

Dna2 endonuclease is also necessary for this step

How do you think the PCNA is removed after ligation?

PCNA interacts with RF-C, pol , Fen1, DNA ligase,CAF1 and MCMT

Several of these have a common motifused in the interaction: Q-X-X-L/I/M-X-X-F-F/Y

p21/CIP1/WAF1, a protein induced by the tumor suppressor p53 uses the same motifto interact with PCNA

What effect is p21 expected to have on DNA replication/repair?

FEN1 interacts with PCNA

Prokaryotic Function Eukaryotic

SSB ss DNA coating RPA

PRIMASE RNA primer synth. Primase subunitof DNA POL ,

5'-3' polymerase DNA POL ,DNA POL

POL III CORE 3'-5' exonuclease DNA POL 5'-3' exonuclease Fen1

COMPLEX ATP dependentclamploader

RF-C

CLAMP processivity factor PCNA

LIGASE seal nicks Lig1

DnaB Helicase ?

ELONGATION FACTORS ARE CONSERVED

DNA Helicase

* Helicase binds ss DNA

* Hydrolyzes ATP to move alongssDNA and peel of substrate DNA

* Can move 5‘ to 3’ or 3‘to 5’ or in both directions on ssDNAand is classified as such

* Dimers or hexamers

P P

5‘ 3‘

3‘ 5‘ 5‘3‘

Substra

te

Boiled su

bstra

te

+he

licas

e

+helic

ase+

ATP

15mer 30mer

15mer30mer

Conserved helicase motifs:

Includes the following for ATPbinding and hydrolysisI = Walker A motif GxGxGKTII = Walker B motif DEad

Binds ATP

Tight conformation

Hydrolyzes ATP

Relaxed conformation

Helicases are ATP driven molecular motors

Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes)

Yeast containing Leu2 on a plasmid

Plate yeast on Leucine minus plates to estimate rate of plasmid loss

Grow under non-selective conditions

MCM8

MCM9

Form hexamer

Associates with MCM2-7

Newbies of unknown function

MCM homolog fromM. thermoautotrophicum(an archaebacterium)has helicase activity.

What direction?

Mt MCM formsdouble hexamers

T antigen helicase domain: XS Chen, Nature, 2003;423:512-8

MtMCM-N terminal (non-helicase) domain: XS Chen, Nature Str. Biol. 2003;10:160-7

Model of replicative double-hexameric helicase action : XS Chen, Nature, 2003;423:512-8

Formation of pre-Replicative complex

Annual Review of Biochemistry2002. Bell and Dutta

G2M

ORC

MCM MCM

CDC6 +Cdt1

Replication Machinery

“Pre-Replicative Complex”

Replication complexes during the cell cycle

ORC

CDC6+Cdt1

Slate G1

early G1

Eukaryotic initiation complex

ORC : A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size, with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity. 

CDC6/Cdc18 : An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1- S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis, making it a critical regulator of DNA replication. 

MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem to function together in a large multi-subunit protein complex.MCM 2-7 is most likely the replicative DNA helicase. 

A/T DUEOREAuxiliaryelements

Auxiliaryelements

Origin recognitionproteins

TranscriptionFactors

TranscriptionFactors

Leading str

Leading str

Lagging str

Lagging str

Core origin

Origins are multi-partite

The Structure of ARS1(S. cerevisiae)

B1 ACSB2B3

ABF1

ORC

“Post-RC” protection

“Pre-RC” protection

13

456

4 1

22

ORC uses different strategies for binding DNAin different species

Eukaryotic initiation complex

ORC : A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size, with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity. 

CDC6/Cdc18 : An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1- S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis, making it a critical regulator of DNA replication. 

MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem to function together in a large multi-subunit protein complex.MCM 2-7 is most likely the replicative DNA helicase. 

The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts Made

from Xenopus egg extracts

Rxn Time:0-30 min30-60 min60-90 min

+ + +

+ + +

+ + +

+ + +

32P labeledDNAreplicationproduct

Replication initiation required ondouble-stranded DNA substrate

Only elongation required onsingle-stranded DNA substrate

Orc is required to load CDC6;CDC6 is required to load MCM

Immunostaining for CDC6 can pick outproliferating cells in Pap smears

Eukaryotic initiation complex

ORC : A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size, with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity. 

CDC6/Cdc18 : An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1- S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis, making it a critical regulator of DNA replication. 

MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem to function together in a large multi-subunit protein complex.MCM 2-7 is most likely the replicative DNA helicase. 

Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes)

Yeast containing Leu2 on a plasmid

Plate yeast on Leucine minus plates to estimate rate of plasmid loss

Grow under non-selective conditions

MCM8

MCM9

Form hexamer

Associates with MCM2-7

Newbies of unknown function

Propidium iodide staining

No

. o

f ce

lls

Fluorescence analysis and cell sorting (FACS)

DNA in cells stained with propidium iodide.Intensity of staining in each cell is proportional to amount of DNA in the cell.

Cross-link protein-DNA I.P. with anti MCM antibody

Reverse cross-link Purify DNA in I.P.

PCR to detect if your sequence was I.P.ed

MCM

anti-MCM Ab

Chromatinimmunoprecipitation

(ChIP) tests whether a given DNA sequence is bound by a protein

in vivo

Role of Pre-RC in mammalian cells

ORC :CDC6/Cdc18 : Cdt1:MCM2-7 :

Human ORC

Quintana, 1997, 1998Pinto/Quintana, 1999Thome 2000Dhar, 2000, 2001

ORC4

Wild type cells /- cells

Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs cells

Cell proliferation of ORC2 and cells measured by MTT assay

oriP

EB

NA

1

EBNA1 protein

Replication from an origin in Epstein-Barr virus:a plasmid expressing EBNA1 and containing oriP will

replicate and be maintained as an episome inmammalian cells

Hygromycin-Resistance

gene

Establishment of drug-resistant coloniesafter transfection of EBV-based plasmid

m

m

mm

m

m

DpnI

DpnI

DpnI

DpnI

m

m

m

DpnI

DpnI

X

X

X

DpnI

DpnI

DpnIX

X

X

Bacterial plasmidMethylatedDpnI susceptible

Replicated once inhuman cellsHemi-methylatedDpnI resistant

Replicated twice inhuman cellsUnmethylatedDpnI resistant

DpnI-resistance assays episome replication in mammalian cells

1)EBV-based plasmid replication is decreased in /- cells. 2) Rescued by plasmid expressing ORC2

+ +

Some cancers have an Achilles Heel

Viral episomes that carry viral oncogenesAmplicons of cellular oncogenes, or of drug resistance genes

Normal Cancer