L4 Flashcards
How are large eukaryotic chromosomes duplicated?
From multiple DNA replication origins that act in a bi-directional manner
How many replication origins are on yeast chromosomes?
400/500
Can only initiate once per cycle
Yeast replication origins
Allow plasmids to be stabile maintained in yeast cells - Autonomously Replicating Sequences (ARS)
Contains 4 regions - A, B1, B2, and B3
Structure of replication origins
Contains 4 regions - A, B1, B2, and B3
Domain A which is the critical DNA sequence - 11bp
ACS sequence of domain A is the key to allow initiation of replication
Orientation of domain A is crucial
5’ and 3’ flanking domains important for function
What does the AT rich sequences around the ACS indicate?
Indicates it may be involved in DNA unwinding
Less H bonds so easier to separate strands
Lowers energy needed for separation
How can B1 be flipped from different origins even though theres no homology?
Sequences are interchangeable
B1 can be replaced by other B1s
But you can’t replace B1 with B2
Can swap B1 domains between different ARS elements - not interchangeable within an ARS element
What does the B3 sequence do?
Binds the Abf1 protein
Connection between transcription and replication thats not completely understood yet
What does the ORC do?
Origin Replication Complex
Binds to the ACS region of domain A which is critical for replication
Roles of the domains
Flanking regions unknown but probably involved in unwinding
Domain A specifically binds components of the initiator complex
Proteins that bind the ARS element
Abf1 transcription factor
6 protein subunit complex called the ORC binds binds domain A and part of B1 in an ATP dependent manner
Mutations of domain A that affect function in plasmid assays weaken ORC binding
ORC binds domain A throughout the cell cycle hence can’t be sufficient for DNA replication
How many ORCs are there?
Orc1 Orc2 Orc3 Orc4 Orc5 Orc6
Initiation of DNA replication in yeast
2 step process
1) At the end of mitosis, formation of the PRC over the origin which involves association of ORC with Cdc6 and coincides with nuclear entry of Mcm proteins
2) Cells must pass through START to initiate DNA replication
What is the licensing factor model?
Diffusible DNA replication inducing factor that is inactivated following DNA replication ensuring that DNA replication only occurs once per cell cycle
This inducing factor gains access to the nucleus following mitosis this ensuring that DNA replication can only reinitiate once mitosis is complete
What is a licensing factor?
A protein or complex of proteins that allows an origin of replication to begin DNA replication at that site
What did studies of HELA cells show about replication?
They took HELA cells in S phase and fused them with another HELA cell in G1 phase and looked to see what happened in the nuclei at replication
– They found that the cell nucleus in S phase wasn’t affected & went on as normal
– Nucleus in G1 went into replication a lot quicker than it normally would’ve done
What if you take a cell in G2 and fuse it to a cell in S phase??
– G2 nucleus - replication didn’t occur
– Only difference between G1 and G2 is that it’s gone through S phase
Something happens at S phase that that prevents it being initiated by the S phase cell
How does the S phase cell initiate replication in the G1 cell??
– Created the term the licensing factor model
– Idea was that whatever the inducing factor in S phase was, maybe it gains access to the nucleus following mitosis (when the cell is in G1)
– DNA replication only occurs when mitosis has finished – proteins disappear and they can’t get access to the nucleus until mitosis is complete
Nuclear membrane breaks down after mitosis – does the nuclear membrane acts as a barrier to the G2 cell stimulating replicating??
– They made the nuclear membrane in a HELA cell permeable in G2 and fused it with the S phase cell
– This time it underwent replication again even through it was in G2 – disaster for the cell
