L5 Flashcards
(26 cards)
Why is the PRC alone not sufficient for initiation of DNA replication?
Also need:
• Activation of Cdc28/Clb kinase
• Activation of Cdc7/Dbf4 kinase
• Cells must pass START to initiate DNA replication
What are the G1 phase cyclins?
Cln1
Cln2
Cln3
What are the S phase cyclins?
Clb5
Clb6
What are the G2 phase cyclins?
Clb1
Clb2
Clb3
Clb4
What happens if you delete all 4 G2 phase cyclin genes?
clb1∆
clb2∆
clb3∆
clb4∆
Cells proceed through START, bud and synthesise DNA
Clb1,2,3&4 are not needed for budding or replication
What happens if you delete both S phase cyclin genes?
clb5∆
clb6∆
DNA synthesis but not bud emergence
DNA synthesis is delayed
They are therefore somehow involved in the initiation process
What happens if you delete both the G2 and the S phase cyclin genes?
Multiple buds
No DNA synthesis
The G1 cyclins are still present but still no DNA synthesis
Suggests that the Clb cyclins are responsible for DNA synthesis not the G1 cyclins
G1 cyclins are not essential for replication
Biochemical & genetic evidence that Clb5 and Clb6 are involved in S phase
Activity of Cdc28/Clb5 and Cdc28/Clb6 kinases peaks at beginning of S phase
Activity of the other Cdc28/Clb kinases peaks much later
Clb5 mutants have a protracted S phase
Clb5 & Clb6 are important for the initiation of DNA replication & other Clbs have a latent ability to promote S phase
What are the 2 possible mechanisms for the regulation of Cdc7/Dbf4
Regulation by Dbf4
Regulation by phosphorylation
Regulation of Cdc7/Dbf4 by Dbf4
Dbf4 = dumbbell formation - how the cells look down a microscope
Experiments demonstrated that Cdc7 protein required an interaction with Dbf4 suggesting that Dbf4 is a cell cycle specific regulatory subunit of Cdc7
Although Dbf4 has no homology to cyclins, it appears to be somewhat analogous in function to cyclins
Regulation of Cdc7/Dbf4 by phosphorylation
Cdc7 is phosphorylated
Hence another regulatory mechanism of Cdc7 activity is phosphorylation
What are 2 hybrid experiments?
Molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions
Interaction = blue colonies
Role of Cdc28/G1 cyclins for initiation of DNA replication
Activate Cdc7/Dbf4 activity through expression of Dbf4.
Activate Cdc28/Clb5 and Cdc28/Clb6 by activating expression of CLB5 and CLB6.
Target the Sic1 protein, an inhibitor of Cdc28/Clb kinase for degradation
Why is it important that the Sic1 inhibitor is degraded?
Cdc28/G1 target the Sic1 for degradation
Cdc28/Clb5 is critical for replication so we need to get rid of the inhibitor
What happens when you remove all G1 cyclins?
The cell arrests in G1
What happens when you remove all G1 cyclins as well as Sic1?
Quadruple mutant
Cell no longer arrests - grows badly but is still alive
Suggests that the main role of the G1 cyclins is to get rid of the inhibitor
Is initiation of DNA replication regulated by the expression of components of the DNA replication machinery?
In S. cerevisiae many components of the replication machinery are regulated by MBF
However, most components of DNA replication machinery are not rate limiting
MBF mutants have a slower S phase but are not blocked for DNA replication
Hence regulation of gene expression of replication machinery components is probably not a key regulatory step
Components of replication machinery are conserved – natural selection is acting on it
Regulation of S phase by G1 protein kinases
When you activate START you activate the Cdc28-Cln3 complex which activates 2 transcription factors: SBF and MBF
SBF regulates CLN1/2 expression
MBF regulates DBF4 and CLB5/6 expression
What does CLN1/2 do to regulate S phase?
CLN1 and CLN2 regulate via the activity of the proteins Cdc4, Cdc34 & Cdc53
The kinase complex inhibits Sic1
By inhibiting Sic1 you are reliving the inhibition of the Cdc28-Clb5/6 complex
What does CLN5/6 do to regulate S phase?
You need Clb5 and Clb6 to get activation of the Cdc28 kinase
Cdc28-Clb5/6 interacts and regulates the PRC
What does DBF4 do to regulate S phase?
You need Dbf4 to get activation of Cdc7
Cdc7-Dbf4 activates the pre-RC
Why is the decision to activate replication a multi-step process?
Gives the cell the option to feed in many different sensing mechanisms
Only when all the conditions are right, the cell can enter replication
Layers multiple essential decision-making processes
YOU ONLY WANT THIS TO HAPPEN ONCE
How is re-replication prevented in S phase?
Evidence that high Cdc28/Clb kinase prevents formation of the pre-RC essential for initiation of DNA replication
Cdc28/Clb activity drops after mitosis hence not only licencing model but lowering Cdc28/Clb may be essential for the formation of the pre-RC
Cdc28/Clb may prevent pre-RC formation by targeting proteins for degradation
Mutations of cdc16 and cdc27, encoding subunits of proteolysis machinery, undergo multiple rounds of DNA replication with no mitosis
How is the PRC inactivated following initiation of DNA synthesis?
After you initiate replication, the activity of the 2 proteolysis elements (cdc16 and cdc27) leads to the destruction of proteins such as cdc6
This leaves ORC binding the DNA
This is an example of an interlocked decision – means the cell can’t do the wrong thing when it’s doing the right thing
