DNA Replication ORC anneals to origin ORC recruits MCM MCM recruits Cdc45p Cdc45p recruits pol  /primase complex RFC displaces pol  and recruits PCNA PCNA recruits pol  DNA ligase stitches DNA fragments together

Regulation of replication Once and only once –Licensing –DNA damage Coordinated with cell cycle –Cyclin –Cyclin dependent kinase (CDK) –Cyclin kinase inhibitor (CKI)

Key regulatory proteins cdc6/cdt1: licensing agents E2F/DP1: S-phase transcription factor Retinoblastoma: E2F repressor p53: cell cycle withdrawal transcription factor p27/p21 KIP: cyclin kinase inhibitors

Cell Cycle G 1 S –Replication G 2 M –Division G 0 –Terminal differentiation

Cell cycle control Cyclins –Cell cycle regulated proteins Cyclin dependent kinases (CDK) –Signaling effectors Cyclin kinase inhibitors (CKI)

Checkpoint regulation Phase progression tied to successful completion of prior phase –ALL DNA healthy –ALL DNA replicated –ALL DNA attached to mitotic spindles Negative/inhibitory regulation –Signal-to-noise –Presence of “No-Go” signal –Threshold of “Ready” signal

Assembly of preRC ORC, cdc6, MCM/cdt1 Immediately following mitosis cdt1 –Recruits MCM –Inhibited by geminin cdc6 –Inhibits MCM helicase –Translocates to cytoplasm CyclinA/CDK2 disrupts MCM inhibition in S

Initiation of replication Cyclin A/cdk2 Releases ORC inhibition Prevents ORC Re-reformation CyA CDK2 cdt1 ORCMCM cdc6 ORCMCM cdc6 cdc45

Licensing ORC+cdc6 is required to recruit MCM ORC-cdc6 is required to activate MCM Cdc6/Cdt1 “licenses” an ORC for replication

Licensing agents Geminin –cdt1 binding protein –Cell cycle dependent expression cdt1 –Inhibited by geminin –Stabilized by geminin –Phosphorylated by CyclinA/CDK2 in S –Phospho-form is exported & degraded –Removal allows binding of cdc45

DNA Damage Base mismatches Single strand breaks Double strand breaks Oxidation/nitrosylation

Strand Break Non-homologous end joining –Ku mediated recognition of ssDNA –End-to-end repair Homologous recombination –Rad51 mediated search for homologous template –Template derived patch Ku Rad51 NHEJ HR

Strand Break ATM kinase recruited to strand break –Ataxia-Telangiectasia Mutated kinase –Autophosphorylates –Phosphorylates H2A –Phosphorylates p53 p53 –Stabilized and activated by phosphorylation –Activates p21 waf/cip (cdk inhibitor) –Blocks transcription of Pol  –Blocks transcription of CyclinA

Determination to divide Integrative Environmental cues Systemic/hormonal controls Internal program

G1 progression E2F/DP1 transcription factor –DNA polymerase –Cyclins A & E –CDK1 Retinoblastoma (Rb) –De-phosphorylation dependent E2F binding –Represses E2F/DP1 –Protoconogene P53 transcription factor –p21 CKI, MDM2

G1 progression Growth factors cause Rb phosphorylation, which gets degraded, allowing xscription of S-phase proteins Cdk4/CyD phosphorylates Rb… Cdk2/CyE phosphorylate RB…. Cdk2/CyE inhibit p27kip, which inhibits cdk2 ATM activates p53, which leads to transcription of CKIs p21 & p27

p53/Rb Active inhibition of cell cycle Cyclin D CDK 4/6 RbE2F Cyclin A/E Pol a CDK1 DNA Damage ATM kinase p53p21 CIP/WAF CDK2/4/6 Mitogens S-Phase Cycle Progression cdc14 phosphatase Phosphorylates to block binding Binds to block transcriptional activity Promotes transcription Phosphorylates to stabilize & activate Dephosphorylates to destabilize & inactivate Promotes transcription Inhibits activity

Regulatory features CDKs regulate cell cycle –Phase specific transcription –Cyclin E/cdk2 promotes Cdc6 transcription –Cyclin A/CDK2 activates synthesis –Cyclin B/cdc2 deactivate Mcm Rb keeps the gate at G1 restriction –Represses CDK2 & polymerase expression p53 blocks cell cycle & promotes apoptosis –Promotes expression of CKIs

Controls on DNA replication Growth factors/mitogens –Rb phosphorylation –Cyclin D upregulation Nutrient availability –Cell size – amino acids, PO 4 –GSK inactivates cyclinD Stress states –DNA damage –ATM phosphorylates p53  CKI expression