Week 2- DNA replication machinery

Question 1

role of finger domain alpha helix in primer 3 OH end/dNTP interaction
- hints
alpha phosphate
- nucleophilic attack
- pyrophosphate

Answer 1

• finger alpha helix closes around incoming dNTP— allows Oxygen of 3 Oh primer to nucleophilic attack alpha triphosphate of dNTP.
  — leads to release PYROPHOSPHATE

Question 2

role of prokaryotic DNA POLYMERASE III
- core subunits?
- elongation on which strands

Answer 2

• alpha– DNA synth activity
• epsilon– 3-5 exonuclease activity
• theta- subunit
• responsible for strand elongation at both leading/lagging strands

Question 3

role of prokaryotic DNA POLY I
- subunits/activity
- on which strands

Answer 3

• capable of of 5-3 exonuclease activity– facilitates primer removal
• 3-5 exonuclease activity on leading strand– proof-reading
• nucleotide polymerizing domain

found on both lead/lag strands

Question 4

why so many potential origins in genome?

Answer 4

• different cells use different sets of origins
• back up origins available in case primary origins fail

Question 5

Eukaryotic DNA poylmerases and their
- functions/subunits
- location of action

Answer 5

DNA Poly 1 alpha
- part of the 4 SU primase complex
- polymerase activity– synthesizes initiator DNA (lead, laggin strands) which is used for elongation

DNA POL III delta
- lagging strand synthesis
– has polymerase activity SI/ 3-5 exoncl. activity– proofreading
— complexed w/ FEN1 enzyme— lead/lag primer removal

DNA POL II epsilon
- leading strand synthesis
- polymerase activity/3-5 exonucle. activity

Question 6

Role of DNA pol delta (III) - coupled flap endonuclease (FEN1) enzyme

Answer 6

– binds to PCNA (polymerase clamp) through which it removes primers via nuclease activity

Question 7

BONUS: which DNA polymerase extends initiator DNA (made by DNA poly alpha) at the LEADING STRAND?

Answer 7

trick Q— both DNA poly delta (III), and epsilon (II)

REMEMBER: DNA polymerase II is main poly at leading synth

Question 8

Role of DNA ligase

Answer 8

catalyzes a phosphodiester bond between adjacent okazaki fragments— H bond between 3 OH and 5 P

Question 9

describe the prok/euk mechanisms to increase DNA polymerase processivity (i.e. keeps DNAP associated with template)

• describe similarities/differences between prok,euk clamps. Are they homologous

Answer 9

PROK
- beta SU clamp addition increases DNA poly III processivity
Beta SU functions as a dimer of trimers

EUK
- PCNA clamp functions as trimer of dimers–

SIMILARITIES of PCNA, beta clamp
- form hexamer ring around dsDNA and slide down dsDNA

not structurally homologous

Question 10

describe prok/euk clamp loading mechanism

NOTE: both form notch rings around dsDNA– which is released upon DNA association

Answer 10

PROK
- 2 beta subunit clamps are loaded onto dsDNA via gamma SU complex in ATP dependent manner

EUK
PCNA process factor loading via RFC

Question 11

why is DNA replication at end of lagging strand an issue?
- consequences?
- how to resolve

Answer 11

• removal of final primer will leave an short stretch of replicated DNA
• DNA polymerase– no de novo DNA synthesis capabilites
  — may lead to strand SHORTENING– bad

solution— TELOMERES
- specialized sequences at termini
—-tandem repeats which are resynthesized (if lost) by telomerase to achieve 3 prime extension.

Question 12

outline function of telomerase and its period of activation

can it still extend telomere if DNA template is not present?

Answer 12

uses telomere tandem repeat seq as template for 3 prime extension at termini
— synthed during G1 phase– active in S phase

– telomerase has reverse transcriptase activity so it can use PRIMER to synth/extend DNA so YES IT CAN