BIO 404/504 – Molecular GeneticsSlide 2Cdk’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’sSlide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Zou & Stillman AssaysSlide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Overall ConclusionSlide 20Slide 21BIO 404/504 – Molecular Genetics Dr. BerezneyLecture 2: Regulation ofEukaryotic DNA ReplicationRole of cdks (cyclin dependent kinases) in the regulation of the cell cycle and proliferation [Cell Cycle Specific cdks]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’sRegulation of DNA Replication by CdK PathwayLate M/ early G1DeP of pre RC & assembly on replication originsPre RCpre ICAssembly of cdc45 & RPA; Temporal programming over S-Phase Zou & Stillman 2000Early G1Late G1ReplicationG1/ SG1 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 RCOrigin sequenceOrigin recognition complex: 6 ORC’s CDC6 + MCMsZou & 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 transfectionFig 1: Cdc45 associates with Mcm2, pol Є, and RPA 70 in vivoFig 2: Cdc45 Interacts with Mcm2, Pol Є, RPA 70 & 34 in a Cell Cycle [S-phase] Dependent MannerWild-type cells expressing both PolЄ-HA and Cdc45-myc were synchronized in G1 with α-factor. Cells were collected at indicated times after release.Fig 3: Mcm2/Cdc45/RPA Associations Require Active S-cdk: Effect of Overexpression of Sic1ΔNTp with a Gal promoterFig 4: Temporal-related Associations of Cdc45 with ARS Sequences: ChIP Analysis; Cyclin B EffectFig 5: Effect of cdc7 & dbf4 Mutants on Cdc45 Association with ARS 1 [ 0 & 30 min after release of the α-block]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)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 replicationFig 7Fig