DNA Replication

• ORC anneals to origin• ORC recruits MCM• MCM recruits Cdc45p• Cdc45p recruits pol/primase complex• RFC displaces poland 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

• G1

• S– Replication

• G2

• M– Division

• G0

– 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

cdt1

ORC MCM

cdc6

ORC MCM

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 p21waf/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 DCDK 4/6

Rb E2FCyclin A/EPol aCDK1

DNA DamageATM kinase

p53 p21CIP/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, PO4

– GSK inactivates cyclinD

• Stress states– DNA damage– ATM phosphorylates p53CKI expression