Transcript Cell Cycle Control - Georgia Institute of Technology

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
• 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
p27/p21 KIP: cyclin kinase inhibitors
p53: cell cycle withdrawal transcription
factor
Cell Cycle
• G0
– Terminal differentiation
• G1
• S
– Replication
• G2
• M
– Division
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/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
• Cyclin A/cdk2
• Releases ORC inhibition
• Prevents ORC Re-reformation
cdt1
CyA
CDK2
cdt1
ORC
MCM
cdc6
cdc45
ORC
MCM
cdc6
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
NHEJ
Ku
Rad51
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 Pold
– 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
Phosphorylates to
block binding
Cyclin D
CDK 4/6
Mitogens
Rb
Promotes
transcription Cyclin A/E
E2F
Binds to block
transcriptional
activity
Pol a
CDK1
Promotes
transcription
Inhibits activity
Phosphorylates to
stabilize & p53
p21CIP/WAF
CDK2/4/6
activate
DNA Damage
ATM kinase
cdc14 phosphatase
Dephosphorylates
to destabilize &
inactivate
S-Phase
Cycle
Progression
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 p53CKI expression