DNA Replication

Question 1

What are the 10 requirements of DNA replication?

Answer 1

DNA polymerase
Template DNA strand
Primer with free recessed 3’-OH
dNTPs and Mg2+
Origin of replication
Helicase
DNA gyrase
Single-stranded DNA binding proteins
Primase
DNA ligase

Question 2

What is the chemical mechanism of action for dNTP addition to an existing DNA chain?

Answer 2

The nucleophilic attack of the 3’-OH of the DNA chain with the incoming dNTP.

Question 3

Cleaving what molecule drives the DNA synthesis reaction forward?

Answer 3

Pyrophosphate into two Ps.

Question 4

How many origins of replication do bacteria have?

Answer 4

1

Question 5

The presence of a leading and lagging strand mean that DNA replication is…

Answer 5

Semi-discontinuous.

Question 6

Which strand runs in the same direction as the replication fork?

Answer 6

Leading strand.

Question 7

The 3’ end of the parental DNA helix is the template for which strand in DNA replication?

Answer 7

Leading strand.

Question 8

Newly-exposed parental DNA on the lagging strand is synthesised discontinuously into…

Answer 8

Okazaki fragments.

Question 9

What specific oligonucleotides does DNA polymerase need to start replication?

Answer 9

RNA primers.

Question 10

What does the 3’-5’ exonuclease activity of DNA polymerase accomplish?

Answer 10

Proof-reading - removing mismatched bases.

Question 11

The removal of single-stranded nicks and RNA primers is done by 3’-5’ or 5’-3’ DNA pol exonuclease activity?

Answer 11

5’-3’.

Question 12

If the wrong base was added, how would DNA polymerase ‘proof-read’ this bad replication?

Answer 12

3’-5’ exonuclease activity.

Question 13

In E. coli, what are the 7 proteins required for DNA replication?

Answer 13

1. Helicase
2. Gyrase
3. SSB proteins
4. Primase
5. DNA polymerase III
6. DNA polymerase I
7. DNA ligase

Question 14

What is the protein that replaces RNA primers with DNA?

Answer 14

DNA polymerase I

Question 15

What type II topoisomerase prevents supercoiling ahead of the replication fork?

Answer 15

DNA gyrase

Question 16

What base pairing do we want the initiation of replication site to be rich in?

Answer 16

A-T, due to the 2 H-bonds.

Question 17

What enzyme translocates along DNA, breaking H bonds?

Answer 17

Helicase

Question 18

What coats the separated DNA strands behind the helicase?

Answer 18

Single-strand binding proteins.

Question 19

What two proteins make up the primosome?

Answer 19

Helicase and primase.

Question 20

What are three functions of single-strand binding proteins?

Answer 20

1. Prevents re-annealing.
2. Prevents secondary structures.
3. Protects from nucleases.

Question 21

What is the main component of the replisome?

Answer 21

Two DNA polymerase III enzymes.

Question 22

Why is a looping arrangement necessary for the lagging strand?

Answer 22

The replisome can move towards the fork as a single unit.

Question 23

What are the three parts of DNA polymerase III?

Answer 23

1. Core enzyme.
2. Clamp loader.
3. Sliding clamp.

Question 24

Which part of DNA pol III keeps its enzyme activity ‘processive’?

Answer 24

The sliding clamp.

Question 25

DNA polymerase III has what exonuclease activity?

Answer 25

Only 3'-5'.

Question 26

DNA polymerase I has what exonuclease activity?

Answer 26

3'-5' and 5'-3'.

Question 27

If a DNA polymerase could synthesise DNA and conduct nick translation, would it be DNA pol I or III?

Answer 27

It would be DNA pol I.

Question 28

The terminus region in E. coli DNA is flanked by what 'one-way valves'?

Answer 28

Terminator sites.

Question 29

What protein finishes up DNA replication in E. coli?

Answer 29

The Tus protein.

Question 30

What are three complexities in eukaryotic DNA replication?

Answer 30

1. Different organelles. 2. Different polymerases. 3. Multiple origins.