Lesson 18: DNA Replication 2

Question 1

DNA polymerase requires what

Answer 1

an origin of replication

Question 2

DNA Polymerases:

Answer 2

initiation and mechanism of action

Question 3

where does replication occur at

Answer 3

replication forks

Question 4

what direction can replication from the origin move in

Answer 4

unidirectionally or bidirectionally

Question 5

what direction does DNA synthesis occur in

Answer 5

unidirectional from 5’ to 3’
—> synthesis only occurs in 1 direction - meaning nucleotides can be only added to the 3’ end

Question 6

reaction mechanism for DNA synthesis

Answer 6

(1) each incoming dNTP is positioned by base pairing with the appropriate template nucleotide

(2) a phosphodiester bond is created by nucleophilic attack of the primer-strand 3’ hydroxyl group of the alpha-phosphate on the incoming dNTP

(3) after phosphodiester bond formation then next nucleotide, dATP, is fitted into the template

Question 7

why is Mg2+ required

Answer 7

metal-ion-catalysis
– neutralizing negative charges

Question 8

leading strand

Answer 8

continuous

Question 9

lagging strand

Answer 9

discontinuous

Question 10

okazaki fragemets

Answer 10

short fragments on lagging strand

Question 11

what direction does the leading strand go in

Answer 11

– same direction of the replication fork

Question 12

excision and end of ookazaki fragments

Answer 12

• RNA primers need to be removed
• okazaki fragments need to be ligated
  —— repliacted DNA shouldn’t contain RNA at the end of the process

Question 13

3 traits of DNA polymerase (DNA pol I)

Answer 13

a – 5’ –>3’ synthesis activity

b – 2 exonuclease activities

c – “slow”, low processivity ( # of nucleotides that can be added before DNA pol I dissociates from DNA)

Question 14

exonuclease activity

Answer 14

• synthesis is 5’–>3’ direction therefore proofreading is 3’–>5’

5’–>3’: RNA primer removal (nick translation) –> removes RNA then fills gap with DNA

Question 15

cellular function of exonuclease activity and function of DNA Pol I

Answer 15

remove RNA primer and fill in with dNMP using dNTPs as substrates

Question 16

DNA pol III

Answer 16

• only proofreading exonuclease activity - fast and highly processive
• where a bulk of DNA synthesis comes form

Question 17

components of the replisome: how many poll III enzymes per replication fork

Answer 17

• 2 poll III enzymes

Question 18

components of the replisome: gyrase

Answer 18

(topoisomerase) unwinds the superhelical tension ahead of the fork as replication proceeds

Question 19

components of the replisome: helicase (DnaB protein)

Answer 19

locally “melts” dsDNA —> ssDNA so Pol III can access information in template strands

Question 20

components of the replisome: primosome

Answer 20

enzymatic component of the replisome that synthesizes short RNA primers de novo (from nothing)

Question 21

components of the replisome: DNA ligase

Answer 21

catalyzes the formation of phosphodiester bond (seals in nicks)

Question 22

both leading and lagging strands are copied by how many replisomes

Answer 22

1

Question 23

single-stand binding protein (SSB)

Answer 23

coats the unwound DNA to prevent it from reannealing

Question 24

what directions does the replisome encounter the DNA strands

Answer 24

in both 5’–>3’ and 3’ –> 5’ direction

Question 25

how many errors occur in bacteria for DNA replication

Answer 25

1 mistake every 1000 bp –> rate of 1X10^-3

Question 26

sources of fidelity:

Answer 26

a: dNTP concentrations: cell tightly controls [dNTPs], too high = increased mutation rate
b: 2 step polymerase reaction: bind correct dNTP via AT: GC base pairing –> ensure proper geometry at active site; then incorporatre dNMP

Question 27

correct base pairing leads to

Answer 27

purine: pyrimidine orientation with a consistent spacial geometry

Question 28

incorrect base pairing leads to

Answer 28

misaligned geometry shown functional groups outside the “ correct region”

Question 29

proofreading by Pol I and Pol III

Answer 29

• both DNA pol I and pol III possess 5’–>3’ synthesis activity with an intrinsic error rate
  – therfore, both require a 3’ –> 5’ exonuclease or proofreading function

– the catalytic site for dNMP incorporation is separate from the proofreading sire