W4 Mechanics of DNA Replication

Question 1

Polymerases:

Answer 1

-Enzyme that can synthesise a new DNA strand from a template sequence

Question 2

E.coli DNA polymerases:

Answer 2

-DNA Polymerase 1:
-Repair of damaged DNA + replication
-DNA Polymerase 2:
-Implicated in repair
DNA Polymerase 3:
-Multisubunit enzyme responsible for de
novo synthesis of DNA

Question 3

DNA polymerases:

Answer 3

-Cannot initiate synthesis
-Requires a primer to provide a free 3’-OH end from which they can extend

Question 4

Unwinding the DNA:

Answer 4

-Topoisomerase relaxes supercoiled DNA
-Initiator protein binds DNA
-DNA helicase binds initiator protein, physically unwinds DNA causing it to denature in that region
-As helicase unwinds the DNA single stranded binding protein stabilizes the DNA and prevents it from forming a 2^o structure.

Question 5

Priming DNA synthesis:

Answer 5

Primase binds to helicase and the denatured DNA (Primase + Helicase = primosome)
-Primase is activated by helicase and synthesizes a short RNA primer for initiation of DNA synthesis - This RNA primer provides 3’-OH group so DNA polymerase 3 can add first nucleotide

Question 6

Okasaki fragments:

Answer 6

-Short RNA primers are synthesised on lagging strand (close to replication fork)
-DNA polymerase 3 lengthens the primer moving away from the replication fork and displaces SSB proteins
-After the replication fork has moved another RNA primer is synthesised and elongated

Question 7

Okasaki fragment synthesis:

Answer 7

1) RNA primer copied from DNA
2) DNA polymerase 3 elongates RNA primers with new DNA
3) DNA polymerase 1 removes 5’RNA at end of neighbouring fragment by 5’-3’ exonuclease activity & replaces with DNA
4) DNA ligase joins adjacent fragments