BIO 404/504 – Molecular Genetics

Dr. Berezney

Lecture 2: Regulation of

Eukaryotic DNA Replication

Role 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’s

Regulation of DNA Replication by CdK Pathway

Late M/ early G1

DeP of pre RC & assembly on replication origins

Pre RCpre 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

Zou & Stillman Assays

• Co-IP (Co-immunoprecipitation) “Pull-down Assays”: cell extractAbs 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

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

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 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 promoter

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

Fig 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 replication

Fig 7

Fig 8