Wk2-cell cycle control and regulation Flashcards
how is re-replication prevented
origin licensing is prevented during S phase until cell division is completed
what is the significance of having helicase loading, helicase activation occur at different cell stages?
- helps to ensure no origin can initiate replication more than once
how were S phase promoting factors (cause origin firing/replication) discovered?
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
what was the conclusion of Blowe/Laskey experiments on DNA replication studies in Xenopus laevis?
i.e. comparing interphase extracts to mitosis extracts
at mitosis-G1, breakdown of nuclear envelope renders DNA susceptible to binding of license factors
S phase-DNA replication initiation mediated by origin firing
what is the result of replication fork passage over chromatin-associated licensing factors?
- what does this have to do with distinguishing replicated/unreplicated DNA
- 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
once fork has passed over origin/LFs are released, when does re-licensing occur?
Next M phase
what is the result of ORI licensing during late/G1
- what licensing factors facilitate this?
assembly of replicative helicase (2 MCM hexamers)
- CDC6-ORC complex associated with cdt1 co-loader which recruit 2 MCMs– form inactive helicase
after pre-replicative complex, pre-initiation complex occurs between G1-S.
What are components of pre initiation complex?
what S
- CMG
- cdc45
- MCM
- GINS
-S-CDK - DDK
what components are responsible for activation of pre-initiation complex?
- DDK, S-CDK —S phase promoting factors
- Mcm10 protein
prevention of re-licensing is prevented by regulating (degrading) what components of licensing factors?
- Cdc6
- MCM
- Cdt1
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
cdc6 overexpression due to assembly of pre-repl. complex
- it is RATE-LIMITING
- mutation leads to decreased origin licensing
how does cdc10 (present at G1/S phase) affect re-initiation of licensing
- rate limiter of DNA replication
- cdt1 overexpression leads to re-initiation
- mutation reduces origin firing
individual roles of licensing factors
- ORC
- Cdc6
- Cdt1
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
how does movement of MCM (also license factor) affect ORI licensing
- how does this differ between eukaryotic organisms
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
change in chromatin- MCM association throughout cell cycle (G1/S, S/G2)?
bonus: during S/G2 phase, what is the state of S-CDK
associates during G1/S
– phosphorylation of MCM—> disassociates from chromatin through S/G2 phase
S-CDK ON
Key feature of mitosis that allows re-replication?
Nuclear membrane breakdown
what needs to occur for 2nd MCM hexamer loading at ORI (to form complete pre-replicative complex)
- cdc6,cdt1 disassociation, re-association. ORC remains
- re-association of cdc6,cdt1 loads MCM 2-7 in opposite direction of 1st MCM hexamer
how do we go from pre-replicative to pre-initiative?
i.e. what components are required?
association of cdc45, GINS, S-CDK, DDK
list of S phase promoters (at G1/S) and their function
- CDK(s) /DDK, Mcm10, CMG
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
how does Cdc45 stimulate replicative helicase activity?
i.e. through what interactions (hint: RNAPs)
- interacts withs RNAP delta, epsilon elongating polymerases
result of of CDK mediated pre- replicative complex association with pre-loading complex CMG (GINS, poly epsilon)
formation of pre-initiation complex which includes elongation polymerases
result of DDK phosphorylation of MCM (which is in CMG) on helicase activity?
conformational MCM change— activation of CMG helicase
potential contribution of Mcm10 to CMG activation and lagging ssDNA extrusion from helicase hexamer?
- hint: involves ATP hydrolysis
may promote ATP hydrolysis—> CMG activation
Mcm10–just because it binds strongly to ssDNA — may help with lagging ssDNA extrusion
MCm10 promotes ATP hydrolysis which may trigger CMG activation
knowing this, is CMG assembly ATP dependent process?
NO
