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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
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