Lecture 13: DNA Replication

Question 1

In which phase of the cell cycle does DNA replication occur?

Answer 1

The S phase

Question 2

Which DNA polymerase is involved in DNA replication? Function?

Answer 2

1 and 3

1: repair and recombination, also helps create the phosphate backbone, getting rid of primers in OKAZAKI
3: adds nucleotides to the synthesising strand (MAIN)

Question 3

How many DNA polymerases does E.Coli have, and how many are actually involved in DNA replication?

Answer 3

5; only 2 involved

Question 4

The growing strand: which way?

Answer 4

Bases added to the 3’ end: so grows 5’ to 3’

Question 5

Why is RNA primer needed?

Answer 5

We need at least a small section of double strand to be able to begin DNA replication. RNA can spontaneously replicate itself whereas DNA cannot (needs primer)

Question 6

Differences in Poly 1 and 3; size, subunits, processivity, speed

Answer 6

Size: Poly 3 > Poly 1
Subunits: Poly 3 > Poly 1
Processivity: Poly 3 > Poly 1
Speed: Poly 3 > Poly 1

Question 7

What does “processivity” of polymerase mean?

Answer 7

The number of bases/things it can add on until it falls off; its limit

Question 8

What is proofreading and what is the difference in proofreading of Poly 1 and 3?

Answer 8

Proofreading/EXONUCLEASE: removing nucleotides
BOTH can proofread 3’ to 5’ (backwards)
ONLY POLY 1 can proofread 5’ to 3’ (forwards)

Question 9

Differences in origin of replication of EUK vs PRO?

Answer 9

Eukaryotes have MULTIPLE origins of replication

Question 10

There is an important section in the DNA strand important for replication; what is it?

Answer 10

The section where there are multiple A-T base pairs. These bonds are weak and therefore this section is easy to separate, in preparation for replication.

Question 11

What is a replication fork?

Answer 11

“The point at which DNA strands are separated to allow replication”. The area where replication actually takes place.

Question 12

Name the proteins involved in INITIATION OF REPLICATION

Answer 12

1. DNA A
2. DNA B
3. Primase
4. Single-stranded DNA-binding protein
5. DNA gyrase (topoisomerase)

Question 13

Another name for DNA gyrase?

Answer 13

Topoisomerase 2

Question 14

What is the function of DNA A?

Answer 14

Recognises the origin sequence, gets the whole process started

Question 15

What is the function of DNA B?

Answer 15

Unwinds the DNA (one at each replication fork/end)’ i.e 2

Question 16

What is the function of Primase?

Answer 16

Synthesises RNA primers

Question 17

What is the function of SSBP?

Answer 17

Binds single-stranded DNA; stablises it, keeps it from “annealing”; base pairing

Question 18

What is the function of DNA gyrase?

Answer 18

Relieves torsional strain generated by DNA unwinding. Facilitates in the negative supercoiling of the DNA

Question 19

How many subunits does helicase have?

Answer 19

6

Question 20

What does helicase do?

Answer 20

It moves along the DNA strand, rotating, and uses ATP to push along to separate the strands; needs energy!!! (ATP)

Question 21

What is an important function specific to DNA polymerase 1?

Answer 21

It can remove nucleotides 5’ to 3’; forwards. It therefore takes out the RNA nucleotides (rNTP) in OKAZAKI fragments and puts in DNA nucleotides (dNTP)

Question 22

What are dNTP and rNTP?

Answer 22

deoxynucleotide DNA) and ribonucleoside tri-phosphate (RNA)

Question 23

What is DNA ligase and what does it do?

Answer 23

Enzyme that “glues” the fragments together, by catalysing the formation of phosphodiester bonds

Question 24

How does the END of a DNA molecule get replicated?

Answer 24

Difficult because no primer; TELOMERASE puts TELOMERES here

Question 25

What ions are an essential part of the catalytic site in DNA polymerase? What amino acid coordinates them and what do the ions do?

Answer 25

Mg2+. Controlled by Aspartic acid. Makes the Mg2+ interact with the phosphate groups on the incoming dNTP

Question 26

When is the exonuclease activity of polymerases used?

Answer 26

In proofreading

Question 27

What is a replication bubble?

Answer 27

The unwinding of the parent DNA from two replication forks that are close to each other, running in opposite directions, forming a ‘bubble’.

Question 28

What does the sliding clamp do?

Answer 28

Keeps the DNA polymerase bound to the DNA strand, so it doesn’t dissociate