DNA polymerase

Question 1

What does DNAP do?

Answer 1

extend DNA by one nucleotide via SN2 reaction
-produces PPi, which is immediately hydrolyed by water to 2Pi

Question 2

What is the structure of DNAP?

Answer 2

polymerase domain (extending DNA!) and exonuclease domain (repair!)
active site: 2 Asp residues with 2 Mg2+ ions to coordinate incoming dNTP

Question 3

Two metal ion mechanism of DNAP

Answer 3

-water acts as base
-3’ OH deprotonated to O:- (stabilised by metal A) can attack alpha phosphate
-e- flow
-pyrophosphate -ve charge (stabilised by metal B)
-pyrophosphate release

Question 4

Three metal ion mechanism of DNAP

Answer 4

3rd metal ion acts as acid -stabilises pyrophosphate

Question 5

How does DNAP select for dNTP over NTP?

dNTP conains deoxyribose -DNA; NTP contains ribose -RNA

Answer 5

steric gate residue
-Phe clashes with OH (found in NTP ribose) but not with H in deoxyribose (dNTP)

Question 6

How is high accuracy achieved by DNAP?

Answer 6

-active site selects for correct base -fingers only close if WC base pair
-steric gate residue selects for correct sugar (clashes if wrong)

Question 7

What happens in DNAP when there is an error in DNA replication?

Answer 7

-DNA primer passes incorrect nt from pol site to exo site
-exonuclease domain removes nt from 3’-5’ (using Tyr as base, 2 metal ions)

Question 8

Specialised translesion synthesis polymerases

Answer 8

-have wide active site to accomodate lesions
-allows replication to conitune (rather than stop completely due to damage)
-only uses 1 metal ions

Question 9

DNAP1 function

Answer 9

Removes primer, fills in gaps on lagging strand

Question 10

DNAP2 function

Answer 10

DNA repair

(DNAP2 is error-prone but fast)

Question 11

DNAP3 function

Answer 11

DNA synthesis
(the primary enzyme!)

Question 12

DNAP4 and 5

Answer 12

allows replication to continue rather than stop when there is damage
(translesion synthesis polymerases)