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DNA Replication. ORC anneals to origin ORC recruits MCM MCM recruits Cdc45p Cdc45p recruits pol a /primase complex RFC displaces pol a and recruits PCNA PCNA recruits pol d DNA ligase stitches DNA fragments together. Regulation of replication. Once and only once Licensing DNA damage

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Dna replication
DNA Replication

  • ORC anneals to origin

  • ORC recruits MCM

  • MCM recruits Cdc45p

  • Cdc45p recruits pola/primase complex

  • RFC displaces pola and recruits PCNA

  • PCNA recruits pold

  • DNA ligase stitches DNA fragments together


Regulation of replication
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
Key regulatory proteins

  • cdc6/cdt1: licensing agents

  • E2F/DP1: S-phase transcription factor

  • Retinoblastoma: E2F repressor

  • p27/p21 KIP: cyclin kinase inhibitors

  • p53: cell cycle withdrawal transcription factor


Cell cycle
Cell Cycle

  • G0

    • Terminal differentiation

  • G1

  • S

    • Replication

  • G2

  • M

    • Division


Cell cycle control
Cell cycle control

  • Cyclins

    • Cell cycle regulated proteins

  • Cyclin dependent kinases (CDK)

    • Signaling effectors

  • Cyclin kinase inhibitors (CKI)


Checkpoint regulation
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
Assembly of preRC

  • ORC, cdc6/cdt1, MCM

  • Immediately following mitosis

  • cdt1

    • Recruits MCM

    • Inhibited by geminin

  • cdc6

    • Inhibits MCM helicase

    • Phosphorylated by CyclinA/CDK2 in S

    • Translocates to cytoplasm


Initiation of replication
Initiation of replication

  • Cyclin A/cdk2

  • Releases ORC inhibition

  • Prevents ORC Re-reformation

cdt1

CyA

CDK2

cdt1

cdc45

ORC

MCM

ORC

MCM

cdc6

cdc6


Licensing
Licensing

  • ORC+cdc6 is required to recruit MCM

  • ORC-cdc6 is required to activate MCM

  • Cdc6/Cdt1 “licenses” an ORC for replication


Licensing agents
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
DNA Damage

  • Base mismatches

  • Single strand breaks

  • Double strand breaks

  • Oxidation/nitrosylation


Strand break
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

NHEJ

Rad51

HR


Strand break1
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 Pold

    • Blocks transcription of CyclinA


Determination to divide
Determination to divide

  • Integrative

  • Environmental cues

  • Systemic/hormonal controls

  • Internal program


G1 progression
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 progression1
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
p53/Rb

  • Active inhibition of cell cycle

Phosphorylates to block binding

Promotes transcription

Cyclin A/E

Pol a

CDK1

Cyclin D

CDK 4/6

Rb

E2F

S-Phase

Binds to block transcriptional activity

Mitogens

Promotes transcription

Inhibits activity

Cycle

Progression

Phosphorylates to stabilize & activate

p53

p21CIP/WAF

CDK2/4/6

DNA Damage

ATM kinase

cdc14 phosphatase

Dephosphorylates to destabilize & inactivate


Regulatory features
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
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 p53CKI expression