Wk2-cell cycle control and regulation

Question 1

how is re-replication prevented

Answer 1

origin licensing is prevented during S phase until cell division is completed

Question 2

what is the significance of having helicase loading, helicase activation occur at different cell stages?

Answer 2

• helps to ensure no origin can initiate replication more than once

Question 3

how were S phase promoting factors (cause origin firing/replication) discovered?

Answer 3

fusion of G1, G2 cells with S phase
G1-S– immediately induced replication
G2-S–no DNA replication– some type of “blocking/prevention” of replication occurring in G2 cell

Question 4

what was the conclusion of Blowe/Laskey experiments on DNA replication studies in Xenopus laevis?

i.e. comparing interphase extracts to mitosis extracts

Answer 4

at mitosis-G1, breakdown of nuclear envelope renders DNA susceptible to binding of license factors

S phase-DNA replication initiation mediated by origin firing

Question 5

what is the result of replication fork passage over chromatin-associated licensing factors?

• what does this have to do with distinguishing replicated/unreplicated DNA

Answer 5

• licensing factors are released after repl. fork has passed/replicated that DNA region

UNREPLICATED DNA– will still have licensing factors, as fork has not passed/removed them

Question 6

once fork has passed over origin/LFs are released, when does re-licensing occur?

Answer 6

Next M phase

Question 7

what is the result of ORI licensing during late/G1
- what licensing factors facilitate this?

Answer 7

assembly of replicative helicase (2 MCM hexamers)
- CDC6-ORC complex associated with cdt1 co-loader which recruit 2 MCMs– form inactive helicase

Question 8

after pre-replicative complex, pre-initiation complex occurs between G1-S.

What are components of pre initiation complex?

what S

Answer 8

• CMG
• cdc45
• MCM
• GINS
  -S-CDK
• DDK

Question 9

what components are responsible for activation of pre-initiation complex?

Answer 9

• DDK, S-CDK —S phase promoting factors
• Mcm10 protein

Question 10

prevention of re-licensing is prevented by regulating (degrading) what components of licensing factors?

Answer 10

• Cdc6
• MCM
• Cdt1

Question 11

how does Cdc6 (present at G1/S boundary) lead to re-initiation of licensing?
- what does this imply about cdc6 and its effect on the rate of DNA replication?
- effect on cdc6 mutation

Answer 11

cdc6 overexpression due to assembly of pre-repl. complex
- it is RATE-LIMITING
- mutation leads to decreased origin licensing

Question 12

how does cdc10 (present at G1/S phase) affect re-initiation of licensing

Answer 12

• rate limiter of DNA replication
• cdt1 overexpression leads to re-initiation
• mutation reduces origin firing

Question 13

individual roles of licensing factors
- ORC
- Cdc6
- Cdt1

Answer 13

ORC– recruited at ORI– acts as scaffold for MCM recruits LFs

CDC6– associates with ORC, forming hexamer ring around dsDNA– loads ORC onto origin DNA

Cdt1– loads MCM2-7 double hexamer at origin via Cdc6-ORC interaction

Question 14

how does movement of MCM (also license factor) affect ORI licensing
- how does this differ between eukaryotic organisms

Answer 14

YEAST
- MCM localizes into nucleus at late M/early G1–> until S phase

MAMMALS
- MCM remains in nucleus throughout cycle

XENPUS
- MCM can freely translocate into nucleus

Question 15

change in chromatin- MCM association throughout cell cycle (G1/S, S/G2)?

bonus: during S/G2 phase, what is the state of S-CDK

Answer 15

associates during G1/S
– phosphorylation of MCM—> disassociates from chromatin through S/G2 phase

S-CDK ON

Question 16

Key feature of mitosis that allows re-replication?

Answer 16

Nuclear membrane breakdown

Question 17

what needs to occur for 2nd MCM hexamer loading at ORI (to form complete pre-replicative complex)

Answer 17

• cdc6,cdt1 disassociation, re-association. ORC remains
• re-association of cdc6,cdt1 loads MCM 2-7 in opposite direction of 1st MCM hexamer

Question 18

how do we go from pre-replicative to pre-initiative?
i.e. what components are required?

Answer 18

association of cdc45, GINS, S-CDK, DDK

Question 19

list of S phase promoters (at G1/S) and their function
- CDK(s) /DDK, Mcm10, CMG

Answer 19

CDK (several) , DDK– via phosphorylating components – recruit cdc45 — which drives assembly of replicative helicase

Mcm10- stabilizes cdc45, GINS association with MCM2-7
- DRIVER OF INITIAL ORIGIN UNWINDING

CMG– complex composed of GINS, MCM, cdc45– pre-Loading complex– formed by CDK

Question 20

how does Cdc45 stimulate replicative helicase activity?
i.e. through what interactions (hint: RNAPs)

Answer 20

• interacts withs RNAP delta, epsilon elongating polymerases

Question 21

result of of CDK mediated pre- replicative complex association with pre-loading complex CMG (GINS, poly epsilon)

Answer 21

formation of pre-initiation complex which includes elongation polymerases

Question 22

result of DDK phosphorylation of MCM (which is in CMG) on helicase activity?

Answer 22

conformational MCM change— activation of CMG helicase

Question 23

potential contribution of Mcm10 to CMG activation and lagging ssDNA extrusion from helicase hexamer?
- hint: involves ATP hydrolysis

Answer 23

may promote ATP hydrolysis—> CMG activation

Mcm10–just because it binds strongly to ssDNA — may help with lagging ssDNA extrusion

Question 24

MCm10 promotes ATP hydrolysis which may trigger CMG activation
knowing this, is CMG assembly ATP dependent process?

Answer 24

NO

Question 25

implication of CMG helicase activation in Euk DNA replication?

Answer 25

known as the committed step

Question 26

upon origin firing and CMG helicase activation, in what direction does MCM move?
- what facilitates further separation?

Answer 26

– INITIALLY– moves in c terminal direction
THEN
- followed by 1st origin melting step— moves N terminal direction

Mcm10/ATP hydrolysis may drive further separation

Question 27

impact of N-terminal first translocation i.e. movement N terminal first?

Answer 27

ensures that DNA at origin is unwound– would not happen if it moved C terminal (immediately away from one another)

Question 28

describe failsafe for bi-directional replication

Answer 28

• Helicases only pass by if both are bound to ssDNA
  — otherwise CMGs would not leave Ori

Question 29

once template exposed, in what order do DNA Polys associate with templates?

what about RFC/PCNA activity

Answer 29

DNAP alpha (primase/polymerase activity containing) — short RNA primer/initiator DNA formation

CDC45 component of CMG recruits elongating polymerases delta, epsilon

RFC loader recognizes 3 prime of initiator DNA– recruits PCNA slide to dsDNA

Question 30

degradation of cdc6 at end of S, cdt1 at S-G2, via various CDKs has what effect on origin licensing

Answer 30

restricts origin licensing to M/G1, origin firing to S

Question 31

Role of Geminin protein on cdt1
role of Cyclin E, A,D/CDK2 on MCM, Cdc6

DNA replication inhibition or activation?
- how can Geminin be exploited in cancer?

Answer 31

• geminin binds cdt1 licensing factor and inhibits it
• cdc6, MCM are phosphorylated by CDKs– displaced from chromatin– activates license origins