Chromosome biology lecture 3

Question 1

Regulating firing of the replication origin

Answer 1

• Ensures DNA replication = only once per cycle
• Involves:
  1. Binding of initiators (ORC) to replicators (ARS/OBRs)
  2. Licensing DNA for replication (assembly of pre-replicative complexes at origins
  3. Assembly of pre-initiation complex (helicase, Mcm2-7)

Question 2

CDK + DDk

Answer 2

• At M/A transition, x cyclins as degraded by APC
• Make pre-RC here
• In late M/early G1, cdc6 made, binds ORC in presence of ATP
• Helps recruit mcm2-7 by ATP hydrolysis, Cdt1 released leaving ORC-CDC6-MCM
• Need another MCM

Question 3

ATP binding to ORC

Answer 3

• If block ATP binding, prevents loading

- Important cancer recognition target

Question 4

Initiation complex forming + firing

Answer 4

• Initiation complex involves loading of replisome components (Cdc25, GINS, cdc6) + firing factors (Skl2, Sld3)
• Firing of replication origin (load DNA pol + other replisome factors, activate IC by phosph)
• Cdc45 recruited to early origins in G1 + late in S phase

Question 5

Firing factors

Answer 5

• Act w/ Dbp11
• If all mutated = lethal so essential
• S-CDK phosph Sld2/3, promotes assoc w/ Dbp11, bind Mcm

Question 6

Switch from initiation to elongation

Answer 6

• 2 forks move away bi-directionally
• Leading strand is cont. w/ PolE, lagging = discontinuous
• CDC45-MCM-GINS stabilises replisome

Question 7

Early + late firing origins

Answer 7

• Context determines if early or late
• E.g. moving efficient early-firing origin to sub-telomeric region where DNA is replicated late confers late replication

Question 8

Affinity for firing factors

Answer 8

• Early origins have ↑ affinity for firing factors, shortage
• Origins in middle of S have ↓ affinity + late S have lowest
• Once fired, firing factors released
• Then bind region of next highest affinity

Question 9

Factors determining origin firing

Answer 9

• Slowing replication speed recruits latest origin
• Licensing factors loaded only in late M/G1, firing factors can be loaded at diff time
• Late origins can be made to fire early (overexposes, ↑ histone acetylation)

Question 10

Late firing origins are actively suppressed

Answer 10

• Late origins x fire if forks stalled/blocks
• Intra-S checkpoint
• Yeast w/ mutated rad53 or ori2-1
• ATM/ATR are kinases
• Prevent replication w/ DNA damage (in yeast, cells treated w/ DNA damaging agent suppress firing, WT cells stop when exposed to y-irradiation, once repaired resume)
• Rad53 inhibits Sld3 by phosph, inhibits DDK

Question 11

Preventing re-replication

Answer 11

• Control MCM loading
• Cdc6 required in late M/early G1
• MCM chaperone cdt1 normally sequestered by geminin
• At M, Gemini is ubiq through APC + degraded → cdt1 chaperone MCM in G1
• Release of cdt1 at loading, PCNA (PIP box)

Question 12

Overexpression of licensing + firing factor (cancer)

Answer 12

• ORC factors ↑ in human cancer, cdc6 particularly ↑ in breast cancer
• MCM dpf4 + cdc7 important in cancer
• MCM staining = better diagnostic for cervical cancer (MCM is expressed in whole epithelial)

Question 13

Oscillation in CDK activity couples mitosis + S phase

Answer 13

• M/A destruction of cyclins → inactivation of Cdk1
• G1/S = Clb kinases active, inactive cdc6, cdc45 loaded onto chromatin in cyclin-cdk dependent reaction, MCM activates
• S phase checkpoint (S,G2, early M)

Question 14

Coordinating progress through S

Answer 14

• Some origins fire early (R bands, exon dense), some late
• Some x act as origins in vivo but replicated passively
• Yeast mutant Clb5/6 = in M assoc w/ Cdk1, M/A transition degrade cyclin Clb + -ve regulatory phosph of Cdk1, G1 = make cyclin but -ve reg. on phosph on Cdk , G1/S -ve re removed by phosphatase