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SV40 ori Li & Kelly SV40 DNA replication In vitro. PNAS (1984) 81:6973 COS cells (infected with SV40) (expresses T Ag) + plasmid + dNTP + rNTP + 32PdATP 37o C, 60 min Analyze products by electrophoresis and autoradiography SV40 + ori + What else could they do to validate their system? Fractionation to homogeneity COS cells (infected with SV40) (expresses T Ag) Human HeLa or 293 cells T Antigen from cell lysate + baculovirus expression system PC 0.2 M KCl 0.66 M KCl (RF-C, pol + pol ) QS 0.2 M KCl 0.4 M KCl (PCNA) ssDNA 0.6 M KCl 1 M KCl RPA ELONGATION FACTORS ARE CONSERVED Prokaryotic Function Eukaryotic SSB ss DNA coating RPA PRIMASE RNA primer synth. Primase subunit of DNA POL , 5'-3' polymerase POL III CORE 3'-5' exonuclease 5'-3' exonuclease DNA POL , DNA POL DNA POL Fen1 COMPLEX ATP dependent clamploader RF-C CLAMP processivity factor PCNA LIGASE seal nicks Lig1 DnaB Helicase ? Sub Gene unit Bacterial Function Eukaryotic dnaE | DNA POL dnaQ (mutD) | POL III CORE | 5'-3' polymerase 3'-5' exonuclease 5'-3' exonuclease | | | COMPLEX | | ATP dependent clamploader dnaX ' dnaX dnaN CLAMP processivity factor DNA POL Fen1 RF-C PCNA CONSERVATION FROM PROKARYOTES TO EUKARYOTES RF-C RF-C 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 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-FF/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 Prokaryotic Function Eukaryotic SSB ss DNA coating RPA PRIMASE RNA primer synth. Primase subunit of DNA POL , 5'-3' polymerase POL III CORE 3'-5' exonuclease 5'-3' exonuclease DNA POL , DNA POL DNA POL Fen1 COMPLEX ATP dependent clamploader RF-C CLAMP processivity factor PCNA LIGASE seal nicks Lig1 DnaB Helicase ? DNA Helicase 15mer 3‘ 5‘ P 5‘ * 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 30mer 3‘ P 5‘ 3‘ te P a T r A st + b e e te u s s a s a tr ca c d i i s l l b e ile e u h h o S + + B * Dimers or hexamers 30mer 15mer Helicases are ATP driven molecular motors Conserved helicase motifs: Includes the following for ATP binding and hydrolysis I = Walker A motif GxGxGKT II = Walker B motif FFFDEad Binds ATP Tight conformation Hydrolyzes ATP Relaxed conformation Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes) Yeast containing Leu2 on a plasmid Grow under non-selective conditions Plate yeast on Leucine minus plates to estimate rate of plasmid loss Form hexamer Associates with MCM2-7 Newbies of unknown function MCM8 MCM9 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 2002. Bell and Dutta Replication complexes during the cell cycle CDC6+ Cdt1 ORC Replication Machinery late G1 CDC6 + Cdt1 MCM S MCM “Pre-Replicative Complex” early G1 G2 M ORC 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 prereplicative 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. Origins are multi-partite Transcription Factors Auxiliary elements Origin recognition proteins A/T ORE Core origin Leading str Lagging str Lagging str Leading str Transcription Factors DUE Auxiliary elements The Structure of ARS1 (S. cerevisiae) ORC 3 6 ABF1 B3 B2 5 22 1 4 1 ACS B1 “Post-RC” protection “Pre-RC” protection 4 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 prereplicative 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 Only elongation required on single-stranded DNA substrate Replication initiation required on double-stranded DNA substrate 32P labeled DNA replication product Rxn Time: 0-30 min 30-60 min 60-90 min + + + + + + + + + + + + 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 prereplicative 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 a plasmid Grow under non-selective conditions Plate yeast on Leucine minus plates to estimate rate of plasmid loss Form hexamer Associates with MCM2-7 Newbies of unknown function MCM8 MCM9 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. Propidium iodide staining MCM Chromatin immunoprecipitation (ChIP) tests whether a given DNA sequence is bound by a protein in vivo Cross-link protein-DNA I.P. with anti MCM antibody anti-MCM Ab Reverse cross-link Purify DNA in I.P. PCR to detect if your sequence was I.P.ed Role of Pre-RC in mammalian cells ORC : CDC6/Cdc18 : Cdt1: MCM2-7 : Human ORC Wild type cells D/- cells ORC4 Quintana, 1997, 1998 Pinto/Quintana, 1999 Thome 2000 Dhar, 2000, 2001 Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs D/- cells Cell proliferation of ORC2 +/+, +/- and D/cells measured by MTT assay 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 EBNA1 protein oriP HygromycinResistance gene Establishment of drug-resistant colonies after transfection of EBV-based plasmid DpnI-resistance assays episome replication in mammalian cells m DpnI X m DpnI DpnI X m DpnI DpnI X m m m DpnI X m m Bacterial plasmid Methylated DpnI susceptible m DpnI DpnI X Replicated once in human cells Hemi-methylated DpnI resistant DpnI X Replicated twice in human cells Unmethylated DpnI resistant 1)EBV-based plasmid replication is decreased in D/- cells. 2) Rescued by plasmid expressing ORC2D + + Some cancers have an Achilles Heel Viral episomes that carry viral oncogenes Amplicons of cellular oncogenes, or of drug resistance genes Normal Cancer