Li & Kelly SV40 DNA replication In vitro. PNAS (1984) 81:6973

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

2. Fractionation to homogeneity

3. ELONGATION FACTORS ARE CONSERVED

9. 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 g complex

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

12. Dna2 endonuclease is also necessary for this step

13. How do you think the PCNA is removed after ligation?

15. PCNA interacts with RF-C, pol d, 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?

16. FEN1 interacts with PCNA

17. ELONGATION FACTORS ARE CONSERVED

18. 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

19. 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

20. Grow under non-selective conditions 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

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

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

23. Mt MCM forms double hexamers

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

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

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

27. Formation of pre-Replicative complex Annual Review of Biochemistry 2002. Bell and Dutta

28. CDC6+ Cdt1 ORC Replication Machinery MCM MCM ORC Replication complexes during the cell cycle CDC6 + Cdt1 late G1 S early G1 “Pre-Replicative Complex” G2 M

29. 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. 

30. Auxiliary elements A/T ORE DUE Auxiliary elements Transcription Factors Transcription Factors Origin recognition proteins Origins are multi-partite Core origin Leading str Lagging str Lagging str Leading str

31. 3 1 2 2 6 ABF1 4 5 4 1 “Post-RC” protection “Pre-RC” protection The Structure of ARS1(S. cerevisiae) ORC ACS B 3 B 1 B 2

33. ORC uses different strategies for binding DNA in different species

34. 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. 

35. 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 + + + + + + + + + + + +

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

38. Immunostaining for CDC6 can pick out proliferating cells in Pap smears

39. 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. 

40. Grow under non-selective conditions 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

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

42. 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. No. of cells Propidium iodide staining

43. Chromatin immunoprecipitation (ChIP) tests whether a given DNA sequence is bound by a protein in vivo

45. Role of Pre-RC in mammalian cells ORC : CDC6/Cdc18 : Cdt1: MCM2-7 :

47. Human ORC D/- cells Wild type cells ORC4 Quintana, 1997, 1998 Pinto/Quintana, 1999 Thome 2000 Dhar, 2000, 2001

48. Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs D/- cells

49. Cell proliferation of ORC2 +/+, +/- and D/- cells measured by MTT assay

50. 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 Hygromycin- Resistance gene EBNA1