DNA replication

Question 1

what does the OR I contain?

Answer 1

lots of A:T content

Question 2

what does preparing the ori in E coli require?

Answer 2

DnaA, DnaB, DnaC, SSB

Question 3

what is the purpose of DnaA?

Answer 3

for the DNA to wrap around it (bindin to consensus seqs) to displace the positive supercoil strain; wrapping denatures DNA; specific binding of DNA to DnaA recruits more DnaA (~20) to bind to the right-half of ori–ATP consuming processes

Question 4

describe helicase assembly

Answer 4

six DnaCs (helpers) binds to six DnaBs to help a hexameric ring of DnaB assemble to each strand; DnaC released and helicase will use ATP to move in a 5’ to 3’ direction–>because enzyme encircles DNA, this reaction and the enzyme activity are highly processive

Question 5

what are the conformations of helicase during its activity?

Answer 5

ATP-bound (extended), ADP-bound (middle), empty (low)

Question 6

primase general info

Answer 6

synthesizes in 5’ to 3’ direction–>like polymerase; attached to helicase (synthesis is opposite the direction of movement)
–helicase is on the other strand, pulling apart 5’ to 3’
–if primase’s rate of synthesis is less than or eq to helicase, we would never get a proper primer
–this is why it’s good to have a couple primers; 2-3 primases are bound to a helicase–>increases processivity
primase will produce a 10-25 nt RNA primer every 1500-2000 nts

Question 7

components of the DNA polymerase III holoenzyme

Answer 7

2 pol III cores; sliding clamp; clamp loader

Question 8

what is the role of the pol III core?

Answer 8

extends the DNA copy from the RNA primer

Question 9

what is the role of the sliding clamp?

Answer 9

prevents pol III from releasing the DNA–increases processivity; works as a dimer

Question 10

what is the role of the clamp loader?

Answer 10

repositions the clamp when loading a new okazaki frag

Question 11

pol III properties

Answer 11

1 in 10 000 nucleotides will be wrongly inserted; of these only 1 in 1000 wrongly inserted nts will NOT be removed by the 3’-5’ exonuclease function; bout 3/100 correct nucleotides are wrongly excised
processivity: falls off about every 15 nt when not bound to sliding clamp; with sliding clamp, falls off about every 500 000; adds on 500 nts per second

Question 12

pol III core general properties

Answer 12

heterotrimer; 3 diff subunits–alpha, epsilon, theta; error checking rate is by equilibrium: it is faster to add proper nts than to remove them, and it is faster to remove improper nts than to add them; the pol core will pause (because it is difficult to add another nt to an improper jutting-out backbone) which exo senses–cuts out

Question 13

subunit alpha of pol III core

Answer 13

the polymerase with 5’-3’ pol

Question 14

subunit epsilon of pol III core

Answer 14

proofread with 3’-5’ exonuclease activity

Question 15

subunit theta of pol III core

Answer 15

stabilized epsilon and increases exo rate

Question 16

how can one e coli replicate in 20 minutes?

Answer 16

e coli will not just replicate it’s own genome, but it will also replicate its “granddaughter” genomes–>multiple rep forks can be found; one ori is pulled apart synthesized; while the original parent genome is still undergoing replication, the daughter genome is beginning to replicate;

Question 17

what removes the primer in prokaryotic replication?

Answer 17

RNAse H or DNA pol I

Question 18

RNAse H general info

Answer 18

H stands for hybrid–can only cleave RNA:DNA base pairing

Question 19

DNA pol I properties

Answer 19

has 5’-3’ polymerase activity, 5’-3’ exo activity, AND 3’-5’ activity; when it runs into RNA primer can nip it out and replace with DNA; found that it was also used as nick translation clean up

Question 20

what is a klenow fragment?

Answer 20

the 5’-3’ poly activity of DNA pol I once the 5’-3’ exo activity is cut off; cleaved by trypsin

Question 21

role of ligase

Answer 21

used to attach two okazaki fragments; in euks, ligase uses NAD while in proks ligase used ATP; AMP takes an oxygen from the phosphorlyated backbone so the OH group can attack the phosphate, thereby joining them