DNA Replication

Question 1

What were the three original models for DNA replication

Answer 1

Conservative
Semi conservative
Dispersive

Question 2

What experiment proved semi conservative replication to be the correct model

Answer 2

Meselson and Stahl (1958)

Question 3

What was the results for the Meleson and Stahl 1958 experiment

Answer 3

The heavy nitrogen used in the experiment was split equally between the two daughter strands during DNA replication.

Question 4

What experiment showed that the replication of circular DNA is bidirectional

Answer 4

Cairn’s experiment

Question 5

What were the results of Cairn’s experiment

Answer 5

DNA was radiolabelled and spread under a photographic emulsion. The radiographies showed two replication forks proving that replication is circular DNA must be bidirectional

Question 6

How many DNA polymerase are there in Ecoli

Answer 6

5

Question 7

What are the differences between the different DNA polymerase

Answer 7

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

Question 8

How does DNA polymerase start to synthesise new DNA

Answer 8

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)

Question 9

How are errors in base pairing found

Answer 9

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

Question 10

What is exonuclease activity

Answer 10

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.

Question 11

How many types of subunits does DNA polymerase III have?

Answer 11

10

Question 12

What do helicase do

Answer 12

They use ATP to unwind the DNA

Question 13

What do topoisomerases do

Answer 13

They relieve the torsional stress caused by unwinding

Question 14

What do DNA binding proteins do

Answer 14

They stabilise each strand and prevent them from coming back together

Question 15

What do primases do

Answer 15

The make primers

Question 16

What do ligases do

Answer 16

They seal nicks in DNA, like those found between Okazaki fragments

Question 17

What is site oriC?

Answer 17

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)

Question 18

What are the binding sites contained in OriC

Answer 18

R sites (R1, R2, R3, R4, R5)
I sites (I1, I2, I3)

Question 19

How is DNA replication initiated

Answer 19

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.

Question 20

How is the leading strand synthesised

Answer 20

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

Question 21

Explain lagging strand synthesis

Answer 21

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

Question 22

How is replication terminated

Answer 22

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