1. BIO 404/504 – Molecular Genetics Dr. Berezney Lecture 2: Regulation of Eukaryotic DNA Replication

2. Role of cdks (cyclin dependent kinases) in the regulation of the cell cycle and proliferation [Cell Cycle Specific cdks]

3. Cdk’s (Cyclin Dependent Protein Kinases) are major cell cycle regulators which are in turn regulated in the cell. Activation: (1) cyclins A  H (2) CAK’s (cdk activating kinases, (e.g., cdk7: thr 160/161–P) Inactivation: (1) wee1- protein kinase: tyr 15-P (2) CKI’s (cdk inhibitors): Inhibit cdk’s by direct binding, e.g., sic1 and S-cdk’s

8. Regulation of DNA Replication by CdK Pathway Late M/ early G1 DeP of pre RC & assembly on replication origins Pre RC  pre IC  Assembly of cdc45 & RPA ; Temporal programming over S-Phase Zou & Stillman 2000 Early G1 Late G1 Replication G1/ S G1 CDKs phosphorylate transcription factors  increased synthesis of S-CDKs (inactive) G1 CDKs phosphorylate inhibitor (CKI) of S-CDKs (sic1)  degradation of sic1 & activation of S-CDKs. S CDKs + cdc7-dbf4 (heterodimer protein kinase) phosphorylates components of pre RC like ORCs & MCMs) leading to activation of initiation (pre IC) Pre RC Origin sequence Origin recognition complex: 6 ORC’s CDC6 + MCMs

11. Zou & Stillman Assays Co-IP (Co-immunoprecipitation) “Pull-down Assays”: cell extract  Abs on beads  spin down beads  SDS-PAGE & Westerns. [Immunobead Separation”] ChIP (Chromatin ImmunoPrecipitation): x-link protein to chromatin in vivo with formaldehyde  shear chromatin to small fragments  IP the sheared chromatin with Ab to protein of interest  from IP isolate DNA  PCR using appropriate primer pairs Chromatin Association Assay: subfractionate nuclei for chromatin  IP both chromatin (pellet) and supernat.  Run SDS-PAGE and Westerns Epitope Tagging of Proteins: HA (hemagglutinin 9 a.a. epitope) and c-myc epitope tagging and in vivo expression following DNA transfection

12. Fig 1: Cdc45 associates with Mcm2, pol Є, and RPA 70 in vivo

13. Fig 2: Cdc45 Interacts with Mcm2, Pol Є, RPA 70 & 34 in a Cell Cycle [S-phase] Dependent Manner Wild-type cells expressing both PolЄ-HA and Cdc45-myc were synchronized in G 1 with α-factor. Cells were collected at indicated times after release.

14. Fig 3: Mcm2/Cdc45/RPA Associations Require Active S-cdk: Effect of Overexpression of Sic1ΔNTp with a Gal promoter

15. Fig 4: Temporal-related Associations of Cdc45 with ARS Sequences: ChIP Analysis; Cyclin B Effect

16. Fig 5: Effect of cdc7 & dbf4 Mutants on Cdc45 Association with ARS 1 [ 0 & 30 min after release of the α-block]

17. Fig 6: ARS 1 Associations with Cdc45 and RPA [both require S-cdk and Cdc7-Dbf4] are Mutually Dependent (cold sensitive cdc45-1 mutant) & rfa2-2 (RPA 34, Ts mutant)

19. Overall Conclusion Cdc45 and RPA assemble with Mcm2 at pre-RC at times consistent with the initiation of DNA replication of specific yeast ARS’s (origin sequences). This assembly requires the activity of both S-cdk and the cdc7-dbf4 heterodimer protein kinase. It is, therefore, concluded that this assemble is part of the process of pre-IC complex formation at origins and activation for DNA replication

20. Fig 7

21. Fig 8