DNA Replication Flashcards
What were the three original models for DNA replication
Conservative
Semi conservative
Dispersive
What experiment proved semi conservative replication to be the correct model
Meselson and Stahl (1958)
What was the results for the Meleson and Stahl 1958 experiment
The heavy nitrogen used in the experiment was split equally between the two daughter strands during DNA replication.
What experiment showed that the replication of circular DNA is bidirectional
Cairn’s experiment
What were the results of Cairn’s experiment
DNA was radiolabelled and spread under a photographic emulsion. The radiographies showed two replication forks proving that replication is circular DNA must be bidirectional
How many DNA polymerase are there in Ecoli
5
What are the differences between the different DNA polymerase
DNA polymerase I: abundant, but has a low processivity. It is the only one that can exonuclease (proof read DNA) in the 5-3 direction
DNA polymerase (II,IV and V): are all involved in DNA repair
DNA polymerase (III): the principle replication polymerase with the highest processivity
How does DNA polymerase start to synthesise new DNA
The direction of synthesis is always 5-3
DNA polymerase must bind to a primer before replication can begin
On the lagging strand lots of short primers are required (Okazaki fragments)
How are errors in base pairing found
DNA polymerase will only include base pairs that compliment its active site (A-T and C-G). Incorrect base pairings do not have the correct geometry and so are excluded from replication until they are repaired
What is exonuclease activity
All DNA polymerases can proof read DNA in the 3-5 direction. The polymerase can remove nucleotides it has just inserted, allowing for errors to be corrected.
DNA polymerase I can also proof read in the 5-3 direction and moves ahead of the other enzymes.
How many types of subunits does DNA polymerase III have?
10
What do helicase do
They use ATP to unwind the DNA
What do topoisomerases do
They relieve the torsional stress caused by unwinding
What do DNA binding proteins do
They stabilise each strand and prevent them from coming back together
What do primases do
The make primers
What do ligases do
They seal nicks in DNA, like those found between Okazaki fragments
What is site oriC?
The site where DNA replication begins in Ecoli
Highly conserved sequence
Contains 5 repeats of a 9bp sequence (R sites) that forms the binding site for the initiator protein DnaA
Contains a thymine rich DNA unwinding element (DUE)
What are the binding sites contained in OriC
R sites (R1, R2, R3, R4, R5)
I sites (I1, I2, I3)
How is DNA replication initiated
DnaA proteins bind to the R and I sites along oriC
The DNA strands then coil around the proteins and form a complex
This complex puts strain on the DUE region causing it to unwind
DnaB proteins (helicase) are activated by DnaC proteins and then bind to the separated strands in the DUE region
The DnaB helicase then move down each strand and unravel the DNA.
How is the leading strand synthesised
Single strand binding proteins (SSBs) prevent the two strands from rejoining
Primase then adds a primer to the strand
DNA pol III then adds nucleotides
Since replication occurs in the same direction as the replication fork, it happens continuously
Explain lagging strand synthesis
Primase adds a primer at the replication fork
DNA pol III then adds the nucleotides
Since a new primer must keep being placed as the strand moves away from the replication fork, the lagging strand is elongated in fragments (Okazaki fragments)
This causes the lagging strand to form a loop where the clamp holds it in place
The primers from each completed Okazaki fragment are removed by DNA pol I (which then fills in the gap left by the primer)
Ligase then joins each fragment together
How is replication terminated
Regions of DNA called Ter(A-F) are binding sites for the protein terminus utilisation sequence (TUS)
TUS blocks DnaB helicase from progressing, stopping the replication fork.
Tus-Ter complexes are asymmetrical and can block progression from one side whilst permitting it from the other
