Li & Kelly SV40 DNA replication In vitro. PNAS (1984) 81:6973 What else could they do to validate their system?

Fractionation to homogeneity

ELONGATION FACTORS ARE CONSERVED

RF-C is a five-subunit complex All subunits are related in sequence and have ATP binding motifs ATP hydrolysis by RF-C is associated with the loading of PCNA RF-C is the functional homolog of the clamp-loader  complex 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 motif used 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 motif to interact with PCNA What effect is p21 expected to have on DNA replication/repair?

FEN1 interacts with PCNA

ELONGATION FACTORS ARE CONSERVED

DNA Helicase * Helicase binds ss DNA * Hydrolyzes ATP to move along ssDNA and peel of substrate DNA * Can move 5‘ to 3’ or 3‘to 5’ or in both directions on ssDNA and is classified as such * Dimers or hexamers

Conserved helicase motifs: Includes the following for ATP binding and hydrolysis I = Walker A motif GxGxGKT II = Walker B motif  DEad Helicases are ATP driven molecular motors

Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes) Yeast containing Leu2 on aplasmid Plate yeast on Leucine minus plates to estimate rate ofplasmid loss Grow under non-selective conditions

MCM8 MCM9 Form hexamer Associates with MCM2-7 Newbies of unknown function

MCM homolog from M. thermoautotrophicum (an archaebacterium) has helicase activity. What direction?

Mt MCM forms double 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 Biochemistry Bell and Dutta

G2 M ORC 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 MCM2-7 Proteolyzed 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/TDUEORE Auxiliary elements Auxiliary elements Origin recognition proteins Transcription Factors Transcription Factors Leading str Lagging str Core origin Origins are multi-partite

The Structure of ARS1 (S. cerevisiae) B1 ACS B2 B3 ABF1 ORC “Post-RC” protection “Pre-RC” protection

ORC uses different strategies for binding DNA in 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 MCM2-7 Proteolyzed 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 min min min + 32P labeled DNA replication product Replication initiation required on double-stranded DNA substrate Only elongation required on single-stranded DNA substrate

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

Immunostaining for CDC6 can pick out proliferating 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 MCM2-7 Proteolyzed 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 aplasmid Plate yeast on Leucine minus plates to estimate rate ofplasmid loss Grow under non-selective conditions

MCM8 MCM9 Form hexamer Associates with MCM2-7 Newbies of unknown function

Propidium iodide staining No. of cells 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.

Chromatin immunoprecipitation (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, 1998 Pinto/Quintana, 1999 Thome 2000 Dhar, 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 EBNA1 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 in mammalian cells Hygromycin- Resistance gene

Establishment of drug-resistant colonies after transfection of EBV-based plasmid

m m m m m m DpnI m m m X X X X X X Bacterial plasmid Methylated DpnI susceptible Replicated once in human cells Hemi-methylated DpnI resistant Replicated twice in human cells Unmethylated DpnI 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 oncogenes Amplicons of cellular oncogenes, or of drug resistance genes Normal Cancer