7.2 - Chromsome Replication Flashcards
How many origins of replication do bacteria have?
Bacteria have 1 origin of replication (oriC) per circular chromosome
How many origins of replication do Archaea (like eukaruotes) have?
Multiple per chromsome
How is chromsome replication different to DNA replication?
DNA is packaged inside chromosomes so, to completely copy the whole chromosome, multiple DNA replications must occur inside it simultaneously
What steps are needed for chromsome replication?
(1) DnaA-ATP assembly at oriC
(2) Helicase assembly on open DNA
(3) Primase & polymerase + clamp assembly and clamp loader assembly
(4) Rep factory assembly
What is DnaA
The key player in initiating chromosome replication.
Why is DnaA a good target for antibiotics?
All Eubacteria (i.e. bacteria) have DnaA and all of them use it. Since it is only common to bacteria, it is a good target for antibiotics
What are the two forms of DnaA?
(1) The active form: when it is bound to ATP.
(2) The inactive form: when it hydrolyses ATP, and is now bound to ADP
If a cell wants to initiate replication what does it need?
A lot of DnaA-ATP. DnaA-ATP initiates replication by binding to and unwinding an AT region in the DNA at oriC, in the first stage of chromosome replication, and recruiting other proteins to the origin
What does DnaA-ATP bind to?
Repeated 9-mer sequence within oriC (called DnaA box). One oriC has several repeated 9-mer sequences. It binds as a protein helix that wraps around the DNA helix, resulting in the formation of structures that recruit other proteins.
What does the binding of DnaA-ATP lead to?
The separation of strands, using the energy from ATP hydrolysis, at the 13-mer repeat sequence.
What is recruited to to the site of replication after DnaA-ATP binding?
2 hexameric DNA helicases (DnaB) and 2 DNA helicase loaders (DnaC)
What does DnaC do before it is released?
DnaC will open DnaB ring and place the ring around the ssDNA at the oriC. So each single strand has now one DNAB bound around it. Binding of these hexameric helicases represents the commitment step.
What do DNA helicases do (DnaB)?
DNA helicases (DnaB) will use energy from ATP hydrolysis to separate DNA at each replication fork. Each DnaB recruits one DNA primase called DnaG that synthesizes an RNA primer on the strand to which DnaB is bound.
What is used to extend DNA to the right?
The top RNA primer (on the 3’ -> 5’ DNA strand)
What is used to extend DNA to the left?
RNA primer in the bottom (on the 5’ -> 3’ DNA strand)
What is recruited to the DNA template near the first RNA primers?
A complex made of: 2 DNA polymerase III, one clamp loader and one open sliding clamp (DnaN)
How is DnaN loaded onto the DNA template near the RNA primer?
With the help of the clamp loader.
What does the sliding clamp/DnaN do?
The sliding clamp is a protein that serves as a processivity-promoting factor in bacterial DNA replication. It will bind DNA pol III and prevent it from dissociating from the DNA template. DNA sliding clamp also known as \beta subunit of Pol III holoenzyme is encoded by DnaN genes.
What happens once the DNA sliding clamp is properly loaded?
DNA pol III will bind to it and start DNA replication from the 3’ hydroxyl end of the RNA primer, after DNA gyrase unwinds the DNA upstream.
What happens as DNA helicase opens up the replication fork?
The leading strand is continuously replicated whereas the lagging strand is replicated in fragments called Okazaki fragments
What are single stranded bringing proteins? (SSBPs)
As one Okazaki fragment is being synthesized, the un-replicated lagging strand further down in the replisome will be covered with SSBs. SSBs will protect all ssDNA during the process of DNA replication
How does replication on the lagging strand work?
As the lagging-strand DNA polymerase synthesizes one Okazaki fragment, that part of the lagging strand is being looped out (i.e. is forming a loop). When the lagging-strand polymerase bumps into the 5’ end of a previously synthesized Okazaki fragment, the DNA polymerase is released and the DNA clamp is disengaged.
What happens after DNA helicase has moved approximately 1000 bases?
Another RNA primer will be synthesized on the lagging strand, the clamp loader will load another clamp to which DNA polymerase will associate and form a new Okazaki fragment. The lagging strand has several Okazaki fragments.
What are the RNA primers of Okazaki fragments cleaved by?
RNaseH, then DNA pol I uses the 3’ OH group of the Okazaki fragments to fill in the gaps left by RNA primers with DNA nucleotides. DNA Pol III can also remove RNA primers itself but it is less efficient
