1. ATP AMP-PNP

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

3. 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.

4. 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

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

6. 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.

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

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

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

11. Some cancers have an Achilles Heel Viral episomes that carry viral oncogenes Amplicons of cellular oncogenes, or of drug resistance genes Normal Cancer

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

13. Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs  cells

14. Cell proliferation of ORC2  and  cells measured by MTT assay

15. 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

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

17. 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

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

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

21. Transition to replication: from pre-RC to unwinding GINS complex Sld5-Go Psf1-Ichi Psf2-Ni Psf3-San And-1/ CTF4

22. MCM10 : essential function in the initiation of DNA replication in yeast. Required in Xenopus after the loading of Mcm2-7, before the loading of CDC45. In yeast, depletion of MCM10 causes the DNA polymerase alpha, catalytic subunit (p180), to be degraded. In yeast associates with DNA pol alpha p180. In mammalian cells associates with DNA pol alpha p180 and loads it on chromatin through the recruitment of AND1/CTF4 CDC7/DBF4 : A DBF4 dependent kinase (DDK) which, together with Dbf4, is required for the initiation of DNA replication. Possible substrates of this kinase are the MCMs. How do we know this? Cdk/Cyclin: A protein kinase essential for the initiation of DNA replication. Substrates that are activated by Cdk for replication initiation are unclear. MCM, CDC6 and some ORC subunits are phosphorylated, but this phosphorylation inactivates the proteins and prevents re-replication. CDC45 : it seems to activate MCMs to initiate DNA replication. Has a role in the loading of DNA primase/polymerase complex. It and the MCM proteins appear to move along with the replication fork. How do we know this? Transition to Replication

23. GINS : essential function in the initiation of DNA replication in yeast. Required in Xenopus after the loading of Mcm2-7, before the loading of CDC45. Composed of 4 subunits: SLD5, PSF1, PSF2 and PSF3 (the Go-Ichi-Ni-San or GINS complex). Appears to form a ring on EM AND1/CTF4: Discovered in yeast because mutations lead to defects in chromosome-transmission-fidelity. Discovered in mammalian cells as a nuclear protein! But found to co-immunoprecipitate with Mcm10 and with p180 subunit of DNA pol alpha. Required for the stability of p180 and also to load p180 on chromatin at origins of replication. MCM8: Related to MCM2-7 in sequence and has a helicase domain. No yeast homolog. Depletion in Xenopus replication reaction decreases DNA replication by 50% without affecting pre-RC formation. Controversy in human cells, where RNAi of MCM8 has been reported to decrease pre-RC formation. MCM9: Similar to MCM8. Depletion in Xenopus replication reaction decreases pre-RC formation. RNAi in human cells affects cell proliferation. Transition to Replication

24. S phase: Chromosome duplication Mitosis: Chromosome segregation Problems for the cell: (1) Alternation of S and M (2) Completion of S before M and vice versa

25. S and M have to alternate: if not, genomic instability S M G1G1 G2G2 1) cdks prevent licensing or pre-RC formation 2) Any other mechanisms? How does the cell prevent re-replication in the same cycle?

26. With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication? Two observations: When a cell in G1of the cell cycle is fused with a cell in S phase, the DNA of the G1 nucleus begins replicating early. Thus S phase cells have a positive factor for DNA replication that G1 cells lack. When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly- synthesized DNA be replicated again. Thus cells in G2 have a negative factor that disappears in mitosis and/or lack a positive replication licensing factor until they pass through mitosis. Control of Replication through the cell cycle

27. Cyclin/cdk protein kinase (i) yeast with temperature sensitive mutation in CDC28 (a cdk) arrest in G1 (ii) addition of a cdk inhibitor (e.g. p21) to Xenopus egg extracts in G1 inhibits replication DBF4/CDC7 protein kinase (i) ts mutation of CDC7 in yeast blocks cell cycle in G1 (by- passed by a mutation in MCM5) (ii) antibody mediated depletion in Xenopus egg extracts Positive factor present in S phase cells

28. CDC6 (i) degraded in yeasts after G1/S (ii) displaced out of the nucleus after G1/S in mammalian cells MCM2-7 (i) displaced out of the nucleus after G1/S in yeast (ii) loses tight association with chromatin after G1/S in mammalian cells CDC45 (i) loses tight association with chromatin after G1/S in mammalian cells Cdt1 (i) degraded in mammalian cells during S phase. (ii) inactivated by interaction with a protein called geminin. ORC: may be inactivated by phosphorylation Mcm10: degraded during G2 phase Positive factor (replication licensing factor) absent in G2 cells, but present in G1 cells

29. HsCDC6G1S H 2 NCOOH Cy SS Wild-type H 2 NCOOH Cy SS  H 2 NCOOH Cy DD H 2 NCOOH Cy AA Ser  Ala Ser  Asp Proper Subcellular Localization of HsCDC6 is Dependent on Both IntactPhosphorylation Sites and a Functional Cy Motif CDC6 in mammalian cells is exported out of the nucleus In S phase due to phosphorylation by cyclin/cdk G1 S

30. GFP-MCM4 localization through the cell cycle in yeast Movie: http://users.ox.ac.uk/~kearsey/mcm4.html

31. Highly active cyclin/cdk protein kinase (i) inactivated in mitosis due to degradation of cyclins. (ii) artificial inactivation in G2 yeast with ts mutations in the cdk or by induction of a cdk inhibitor results in re-replication of DNA without mitosis. (iii) the nuclear export (in mammalian cells) or proteolysis (in yeast) of CDC6 is due to phosphorylation by cyclin/cdk. Therefore the negative factor present in G2 cells may also be responsible for the absence of a positive replication licensing factor in G2 nuclei! Geminin (i) elevated in G2 cells and degraded in mitosis (ii) addition of a stable form of the protein to Xenopus egg extract inhibits the loading of MCM2-7 on chromatin and prevents DNA replication (iii) interacts with Cdt1. Negative factor present in G2 cells, but absent in G1 cells

32. Discovery of Geminin  Identified in Xenopus as a protein degraded by the Anaphase Promoting Complex during the Metaphase to Anaphase Transition  Inhibits the initiation of DNA replication by blocking the loading of MCM proteins onto replication origins. ORC CDC6 ORC CDC6 MCM Geminin

33. Formation of pre-Replicative complex/ Licensed chromatin Bell and Dutta, ARB 2002

34. Geminin-Cdt1 Geminin interacts with and inhibits Cdt1 High levels of geminin in the latter half of the cell-cycle might prevent re-replication by inhibiting Cdt1 Is geminin an inhibitor of the cell-cycle? A tumor suppressor? Does it really prevent re-replication? Wohlschlegel, Science, 2000

35. Orc2 Cyclin A Cdt1 Geminin Time (hours) 03691221181524 Geminin and Cdt1 Protein Levels During the Cell Cycle

36. Geminin Cdt1

37. Overexpression of Cdt1 and Cdc6 produces cells with > 4n DNA Cell number DNA content BrdU incorporation

38. Geminin Cdt1

39. HCT116(P53 -/- ) Control RNAi Gem4 RNAi 4.8% 53.6% 0.8% 47%

41. S and M have to alternate: if not, genomic instability S M G1G1 G2G2 elevated activity of cdks elevated level of geminin assembly of pre-RC can only occur in a window in G1 (Cdc6 exported, Cdt1 degraded, Mcm2-7 phosphorylated in S)

42. Low cyclin/cdk activity is required at G1/S to initiate DNA replication Low cyclin/cdk activity in G1/S is inadequate to initiate mitosis High cyclin/cdk activity in G2 is required for mitosis High cyclin/cdk activity in G2 inhibits DNA replication Solution to the alternation problem Solution to the completion problem For mitosis to be completed, cyclin-cdk has to be inactivated. Thus S phase cannot occur before mitosis is completed. Why doesn’t mitosis occur before S phase is completed? Checkpoint pathways that are still being worked out.

43. CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC DNA replication checkpoint Spindle checkpoint SG2MetaphaseAnaphase Kinetochore attachment Repair of DNA damage G1 G1 DNA damage checkpoint Cell Cycle Checkpoints p53  p21 S DNA damage checkpoint ATR  Chk1 ATM  Chk2

44. HCT116 contGem4 Phospho Chk1 Phospho Chk2 Chk1 Chk2 Loading control Geminin Loss of geminin leads to re-replication and activation of Chk1 and Chk2

45. Depletion of geminin activates G2/M checkpoint, resulting in sequestration of Cdc25C outside the nucleus (red on right panel: cytoplasmic Cdc25C). Rereplication by depletion of geminin activates the G2/M checkpoint.

46. S and M have to alternate: if not, genomic instability S M G1G1 G2G2 1)elevated activity of cdks 2)elevated level of geminin 3)assembly of pre-RC can only occur in a window in G1 (Cdc6 exported, Cdt1 degraded, Mcm2-7 phosphorylated in S) 4)If despite this re- replication occurs: checkpoint pathways stop the cell-cycle