Week 4 - DNA Replication

Question 1

Meselson and Stahl

Answer 1

experiment proved semi-conservative replication

Question 2

semi-conservative replication experiment

Answer 2

1. heavy DNA is replicated
2. mixed with lighter DNA
   3 2 rounds of replication resulted in HL:LL:LL:HL

Question 3

semi-discontinuous replication

Answer 3

DNA strands ran anti-parallel and the polymerase can only synthesize 5’-3’, therefore there must be a laggin strand

Question 4

enzymes of replication

Answer 4

• nucleases
• polymerases
• helicases
• ligases

Question 5

nucleases

Answer 5

catalyzes hydrolysis of phosphodiester bonds in DNA and RNA

Question 6

nuclease variation

Answer 6

• endo or exo
• RNA or DNA
• ss or ds

Question 7

DNA polymerase

Answer 7

• catalyzes a Nu- attack in 3’OH
• phosphoryl group transfer (releases PPi pyrophosphate, which dissociates into 2, 2Pi)
• requires template, primer, dNTPs
• facilitates Nu- attack through electron withdrawl with the Mg ions

Question 8

dNTP

Answer 8

• synthesizes phosphodiester bond
• “drive” translation
• maintains an overal negative G

Question 9

DNA polymerase reaction mechanism

Answer 9

1. incoming dNTP is attacked at the alpha-phosphate by the 3’OH of the growing chain
2. pyrophosphate is release
3. newly synthesized DNA will base pair with template DNA

Question 10

enzyme cofactors

Answer 10

• 3 Asp residues that form coordination bonds wiwth 2 Mg ions
• in all DNA polymerase
• facilitiates Nu- attack
• one Mg ion facilitates Nu- attack by making alpha-phosphorus the stronger Nu-
• one Mg facilitates release of pyro-phosphate by electron withdrawl from beta and gamma-phosphorus

Question 11

variable features of DNA polymerases

Answer 11

• processivity
• accuracy (fidelity/trueness to template)
• translocation

Question 12

processivity

Answer 12

of dNTPs before the polymerase dissociates from the template (measure of affinity)

Question 13

accuracy/fidelity/trueness to template

Answer 13

• enhanced by proof-reading, substrate-specificity in all active sites, repair of mistakes not fixed by proof-reading
  ex. an E. coli genome DNA pol makes a “mistake” once every 10^9-10^10 insertions

Question 14

translocation

Answer 14

conformation change in DNA polymerase due to release of pyro-phosphate (PPi)

Question 15

3’-5’ exonuclease active site

Answer 15

mispaired base positions in exonuclease site as DNA polymerase will slide back (because it cannot elongate further) and it is removed