DNA Replication

Question 1

What direction does DNA synthesis occur in?

Answer 1

5’ -> 3’ direction

Question 2

What group of a nucleotide is chemically reactive?

Answer 2

3’ OH (hydroxyl) group

Question 3

True or False: Replication occurs in both directions from origin of replication

Answer 3

True

Question 4

Define DNA synthesis on the ‘leading strand’

Answer 4

DNA is continuously synthesised from origin of replication in its 5’-3’ direction

Question 5

Define DNA synthesis on the ‘lagging strand’

Answer 5

DNA is discontinuously synthesised from the origin of replication in its 5’-3’ direction as Okazaki fragments

Question 6

Name the 7 proteins involved in DNA replication

Answer 6

Helicase, topoisomerase, SSBP, primase, DNA Polymerase I and III, and DNA ligase
= honeydew, tomato, strawberry, prune, red and white potato, lettuce

Question 7

What is the main role of the Helicase protein in DNA replication?

Answer 7

Separates the two DNA strands at origin of replication

Question 8

What is the main role of the Topoisomerase protein in DNA replication?

Answer 8

Moves ahead of DNA replication and breaks DNA strands where it has become too tightly twisted, then repairs

Question 9

What is the main role of the Single Stranded DNA Binding Protein (ssbp) in DNA replication?

Answer 9

Keeps the 2 separated strands from rebinding and protects the single strands of DNA from being degraded by other enzymes

Question 10

What is the main role of the Primase protein in DNA replication?

Answer 10

Uses internal 3’ OH group to add a short stretch of RNA primer

Question 11

What is the main role of the DNA Polymerase III protein in DNA replication?

Answer 11

Uses 3’ OH group of RNA strand to begin adding DNA nucleotides that are complementary to parent strand

Question 12

What are the roles of the DNA Pol I protein in DNA replication?

Answer 12

On lagging strand:
- Recognises an RNA/DNA hybrid
- Removes RNA component
- Uses 3’ OH group of adjacent Okazaki fragment to initiate DNA polymerisation where RNA strand was
In general: polymerisation of DNA nucleotides

Question 13

What is the main role of the DNA Ligase protein in DNA replication?

Answer 13

Creates a phosphodiester bond between two Okazaki fragments = one continuous stand of DNA

Question 14

Name the sections of replicated DNA on the lagging strand

Answer 14

Okazaki fragments

Question 15

Describe the joining of 2 replication bubbles.

Answer 15

1. Replication occurs on the leading strand of each bubble, which run in opposite directions towards each other
2. Polymerisation ends at the first RNA section of the next bubble’s lagging strand
3. DNA Pol I recognises the hybrid and replaces RNA nucleotides with DNA
4. DNA Ligase creates a phosphodiester bond between the two segments = continuous strand of DNA

Question 16

What enzyme is involved in progressive addition of new nucleotides?

Answer 16

DNA Polymerase III

Question 17

What is the starting point for nucleotide addition?

Answer 17

Primase enzyme makes RNA primer

Question 18

Which enzyme unwinds/unzips the double helix?

Answer 18

Helicase

Question 19

Which enzyme releases the tension by unwinding the DNA helix?

Answer 19

Topoisomerase

Question 20

Which enzyme prevents the separated strands from rejoining or degrading?

Answer 20

Single-stranded DNA binding protein (ssbd)

Question 21

What replaces RNA nucleotides with DNA?

Answer 21

DNA Polymerase I

Question 22

What enzyme joins fragments of newly synthesised DNA together, and where?

Answer 22

DNA ligase

Lagging and leading strands, within and between replication bubbles.

Question 23

What are the two types of DNA repair from an error?

Answer 23

Endo- and exo- nuclease

Question 24

What is the role of an exonuclease protein?

Answer 24

To removes nucleotides from end of a strand

Question 25

What is the role of an endonuclease protein?

Answer 25

To remove nucleotides from within a strand

Question 26

Which enzyme 'proofreads' the new DNA strands, and how does this mechanism work?

Answer 26

During DNA replication, the DNA Pol III enzyme will check that it has added the right base to correspond to the parent strand, and can remove and replace it if not

Question 27

Does the DNA Pol III enzyme carry out endo- or exo- nuclease activity

Answer 27

exonuclease activity

Question 28

What is the impact of an incorrect base?

Answer 28

Creates a permanent error.

Question 29

What is a permanent error?

Answer 29

When an incorrect base is added, the strand, when used as a template, will reproduce the error in both the new molecule, and in all subsequent strands.

Question 30

What is the purpose of PCR?

Answer 30

To amplify a targeted region of the DNA by creating many copies (rapid exponential increase)

Question 31

Name the 3 main steps in PCR?

Answer 31

- Denaturation - Annealing - Extension

Question 32

What is the purpose and temperature of the denaturation step in PCR?

Answer 32

To seperate the strands of the DNA helix | > 94*

Question 33

What is the purpose and temperature of the annealing step in PCR?

Answer 33

Resynthesized DNA primer binds/anneals to separated strand (by lowering the temp 50-70*)

Question 34

What is the purpose and temperature of the extension step in PCR?

Answer 34

Special DNA polymerases adds nucleotides to strands (at very specific temp = 72*)

Question 35

Name 3 differences between in vivo and in vitro DNA replication

Answer 35

In vivo: - RNA primer that is removed and replaced - 1 leading (continuous) and 1 lagging (discontinuous) strand - Starts at multiple Ori sites - Whole genome - constant temp In vitro: - DNA primer that becomes part of the new strand - 2 continuos strands - Starts at only 2 sites where primer is designed to bind - Replication of only the region between synthesised primers - Cycling of different temps