Lecture 2/5

Question 1

In which direction does replication take place in the cell?

Answer 1

It is bidirectional, with a replication fork on each side of the replication bubble

Question 2

Do prokaryotes have a cell cycle?

Answer 2

No

Question 3

What is the function of topoisomerase?

Answer 3

It relaxes the supercoils of DNA (cuts through) made by helicase

Question 4

Where does topoisomerase cleave DNA? And does it put the DNA back together after?

Answer 4

At the phosphate backbone, and it ligates the strands back together afterward

Question 5

Where do exonucleases work?

Answer 5

At a free end

Question 6

Is topoisomerase an exo or endonuclease?

Answer 6

Endonuclease, it also ligates.

Question 7

What is the order of proteins recruited for DNA replication in prokaryotes?

Answer 7

Initiator protein binds to the origin of replication, recruits helicase to unwind, ssBPs to keep the replication bubble open, primase to make an RNA primer, DNA pol iii for elongation, primers removed by DNA pol i and fragments put together by ligase.

Question 8

How does DNA polymerase elongate on both strands of DNA concurrently when the direction of elongation is in opposite directions?

Answer 8

The DNA polymerase complex twists the lagging strand

Question 9

Where are nucleotides removed from in proofreading?

Answer 9

The 3’ end

Question 10

If there is a mismatch during elongation, the DNA is destabilized in the polymerase active site. How is it fixed?

Answer 10

The DNA is transferred to an exonuclease site, the mismatch is corrected and then flipped back to the polymerase active site

Question 11

If a DNA mismatch is not initially caught by DNA polymerase, can it still be fixed?

Answer 11

Yes, but it will be fixed later and not by polymerase

Question 12

In prokaryotes, does DNA replicate from one site or multiple sites? And is it random?

Answer 12

One site of origin, not random

Question 13

Which is faster, pol i or pol iii?

Answer 13

Pol iii is much faster

Question 14

When is replication finished in a prokaryote?

Answer 14

when the replication forks meet at the termination region and the topoisomerases separate the entwined chromosomes, yielding 2 molecules

Question 15

What is needed for DNA synthesis in a tube?

Answer 15

dNTPs, Mg++ (co-factor), DNA template, DNA primer, polymerase

Question 16

are all 3 DNA polymerases capable of elongation?

Answer 16

yes, in the 5’ to 3’ direction

Question 17

are all 3 DNA polymerases capable of 3’ to 5’ exonuclease activity?

Answer 17

yes

Question 18

what is 3’ to 5’ exonuclease activity?

Answer 18

proofreading

Question 19

are all 3 DNA polymerases capable of 5’ to 3’ exonuclease activity?

Answer 19

no, only pol i

Question 20

what is 5’ to 3’ exonuclease activity?

Answer 20

removal of RNA primers

Question 21

what are the 5 major differences between DNA replication in euks an proks

Answer 21

1. many replication bubbles
2. primase works in complex with DNA pol alpha; elongation with DNA pol delta + PCNA + RF-C (lag) and epsilon + helicase (lead)
3. RNA primers degraded by ribonucleases HI and FEN-1
4. Histone proteins must be dissociated from DNA
5. DNA is ds linear

Question 22

Why do euks have multiple replication bubbles?

Answer 22

the genome is much larger, and replication is much slower than in proks

Question 23

what is the origin site called in euks?

Answer 23

ORC - origin of replication complex

Question 24

how long does it take bacteria to replicate?

Answer 24

20 mins

Question 25

what does primase + dna pol alpha do?

Answer 25

synthesizes the primer

Question 26

which polymerases + proteins are used in elongation in euks?

Answer 26

leading strand: dna pol epsilon + helicase lagging strand: dna pol delta + PCNA + RF-C

Question 27

which replicates faster in euks, leading or lagging strand?

Answer 27

they are the same speed

Question 28

what is used in eukaryotes to unwind and keep the replication bubble open?

Answer 28

helicase unwinds, SSBPs keep it open, and topoisomerases cut the supercoiled DNA and ligate it after to re-seal

Question 29

What does RF-C do?

Answer 29

It binds new DNA strand and recruits PCNA to help load DNA onto polymerases

Question 30

What switch is required for elongation in euks?

Answer 30

the switch from DNA pol alpha to either pol delta (lagging) or pol epsilon (leading)

Question 31

what degrades RNA primers in euks? in proks?

Answer 31

proks: dna pol i euks: ribonucleases HI and FEN-1

Question 32

once HI and FEN-1 remove RNA primers, what fills in the gaps?

Answer 32

DNA pols delta and epsilon, with ligase to catalyze phosphodiester bonds between fragments

Question 33

what happens to histones during euk replication?

Answer 33

they must be removed/dissociated, but are replaced after the replication fork moves through

Question 34

which end of the chromosome experiences shortening?

Answer 34

5', primers are removed but you can't add to the 5' end

Question 35

if there is shortening on one side, what happens on the 3' end? and what do we do about it?

Answer 35

there is an overhang. there are telomeres made of repeats so you're not losing important info

Question 36

what is the Hayflick limit?

Answer 36

the number of times a human cell population divides before stopping

Question 37

what does telomerase do?

Answer 37

it uses an RNA template to lengthen the parent strand

Question 38

What are the 3 steps in the telomerase process?

Answer 38

1. binding of telomerase to 3' overhang, RNA template hybridizes to DNA and provides template 2. elongation, adding bases to parental strand 3. translocation and then more elongation

Question 39

how far does telomerase extend a strand?

Answer 39

50-60 nucleotides

Question 40

is there still an overhang after telomerase adds nts?

Answer 40

yes

Question 41

Why is there no chew back in the chromosome after replication?

Answer 41

the ends are protected by a protein complex called shelterin (which is removed during replication and then re-added)

Question 42

why don't we want chew back on the chromosomes after replication?

Answer 42

it will shorten the chromosome much faster

Question 43

when is telomerase fully turned off?

Answer 43

when a cell no longer divides (senescence)

Question 44

what are the steps of PCR?

Answer 44

1. Denature DNA 2. Primers hybridize to DNA template 3. 20-40 rounds replication