Replication

Question 1

Explain the Meselson-Stahl experiment

Answer 1

Shows that DNA replication is semiconservative using N15 parent and N14 children

Question 2

5 steps of initiation

Answer 2

1. DnaA binds to methylated DnaA boxes
2. DnaA binding relaxes the DUE
3. DnaB binds to open DUE
4. DnaG and Pol III bind
5. Replication commenses

Question 3

DnaA

Answer 3

binds to methylated DnaA boxes to open DUE for replication

Question 4

DnaB

Answer 4

binds to open DUE

Question 5

What is special about the DUE?

Answer 5

It is very A-T rich so it’s weaker from less H bonds

Question 6

DnaB

Answer 6

helicase which binds to DUE

Question 7

SSB

Answer 7

keeps ssDNA from coming back together

Question 8

DnaG

Answer 8

primase

Question 9

Hda1

Answer 9

protein in DNA Pol III that hydrolyzes DnaA-ATP to DnaA-ADP so replication can move forward

Question 10

SeqA

Answer 10

protein that binds hemi-methylated DNA at DnaA boxes to prevent DnaA-ATP rebinding thus preventing origin refiring

Question 11

clamp

Answer 11

sits on the DNA in front of DNA Pol III

Question 12

DNA is synthesized -‘ to -‘

Answer 12

5’ to 3’

Question 13

DNA replication requires what 5 things

Answer 13

1. a template
2. dNTPs
3. a 3’ -OH on the growing strand
4. polymerase
5. Mg2+ cofactor

Question 14

polymerase breathing

Answer 14

if a hairpin loop forms the Pol II lifts up and then settles back down and ends up not transcribing the loop

Question 15

What controls the speed of the replication fork?

Answer 15

DnaG (primase) because lagging strand DNA pol III requires primers to move and is connected to the leading strand DNA Pol III

Question 16

DNA Pol I and RNase H

Answer 16

removes the RNA primer

Question 17

What are the 5 termination steps?

Answer 17

1. Tus proteins slow the replication fork crossing ter sequences
2. Tus proteins are directional and only allow one side to pass
3. Holliday junction formed between tus proteins
4. RecG resolves Holliday junction
5. RecBCD cleaves and ligates

Question 18

RecG

Answer 18

resolves Holliday junctions

Question 19

RecBCD

Answer 19

cleaves Holliday junction extras and ligates ends

Question 20

terC and terA

Answer 20

where termination occurs and Holliday junction formed

Question 21

DNA Pol I

Answer 21

lagging gap RNA primer replacement

Question 22

DnaC

Answer 22

DNA clamp

Question 23

How does E. coli have such a short generation time?

Answer 23

It is already replicating again before the genome is separated

Question 24

Where is oriC in the cell?

Answer 24

attached to the pole

Question 25

How are the chromosomes segregated after replication?

Answer 25

1. parS (site) is bound by parB
2. parS/parB is bound by parA which is tethered to the nucleoid
3. parA is hydrolyzed and passes parS/parB to another parA (rock climbing analogy)

Question 26

PCR requirements

Answer 26

1. template (dsDNA)
2. dNTPs
3. thermophilic DNA pol
4. Mg2+
5. Primers

Question 27

PCR 3 steps

Answer 27

1. denaturing
2. annealing
3. elongation