Lecture 3: DNA Replication

Question 1

Why should DNA be replicated before cells divide?

Answer 1

To ensure transfer of genetic information to progeny cells

Question 2

What happens (consequences) if DNA replication isn’t accurate?

Answer 2

Mutations will occur

Question 3

What are some structural features that aid DNA replication?

Answer 3

• DNA being a stable molecule

• Hydrogen bonds are weak and strands can be relatively easily separated

• Base-pairing rules guide the synthesis of complementary strands

Question 4

What are the basic requirements for DNA replication?

Answer 4

• Deoxy-ribo-nucleoside triphosphates (dNTPs) to create the new strand

• A template to copy from

• A 3’ end to add nucleotides to

• An enzyme to do the polymerisation

• An energy source

Question 5

DNA polymerase operates in 5’ to 3’ OR 3’ to 5’?

Answer 5

5’ to 3’

Question 6

What are the challenges for DNA replication?

Answer 6

• Need to separate strands

• Strands run in opposite directions (anti-parallel 3’-5’ and 5’-3’)

• Need to prevent DNA reannealing

• Need to provide a 3’ -OH group to attach new nucleotides to

Question 7

How does DNA overcome the strand separation challenge?

Answer 7

Helicase in the replication fork opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases

Question 8

Topoisomerase function
(the replication fork)

Answer 8

Helps relieve the strain on DNA when unwinding by causing breaks, and then resealing the DNA.

Breaks the bond & then reseals it.

Question 9

What prevents the DNA from reannealing?

Answer 9

Single-stranded DNA binding proteins.

They bind to the separate strands and help prevent reannealing.

Question 10

What is the difference between Helicase and Topoisomerase?

Answer 10

The main difference between helicase and topoisomerase is that helicase unwinds the double-stranded DNA whereas topoisomerase relieves the tension created by helicase.

Furthermore, helicase breaks the hydrogen bonds between the two DNA strands while topoisomerase breaks the phosphodiester linkages in the DNA backbone.

Question 11

If DNA needs to provide a 3’ -OH to attach new nucleotides when replicating, how does the process of replication even start?

Answer 11

DNA primase makes a short RNA primer that provides the 3’ -OH for DNA polymerase

Question 12

What is DNA primase also known as? What does it mean?

Answer 12

RNA polymerase

It doesn’t need an existing 3’-OH, because RNA predominantly has 1 strand

Question 13

What is an RNA primer?

Answer 13

A short stretch of RNA ~5-10 nucleotides in length complementary to the template of DNA.

• It serves as a starting material for a polymerisation process.

Question 14

What is a semi-conservative strand?

Answer 14

When double helix is unwound and each strand acts as a template for the synthesis of a complementary strand.

Forms two daughter DNAs each consisting of one original strand and one new strand.

Question 15

What provides energy for chain elongation?

Answer 15

Hydrolysis of pyrophosphate

      O              O
     ||             || -O — P — O — P — O-
      |               |
      O-             O-

Question 16

What is a phosphodiester bond?

Answer 16

Bond that forms between phosphate and sugar. (P-O-HC)

Question 17

Helicase function

Answer 17

Opens the DNA helix by breaking down hydrogen bonds between the nitrogenous bases.

Question 18

Why does the antiparallel nature of DNA provides a challenge to DNA replication?

Answer 18

Because DNA polymerase moves in 5’ to 3’ direction, just like one of the DNA strands.

Question 19

Define leading strand

Answer 19

One of the DNA strands that moves in 3’ to 5’ direction.

Question 20

Key feature of leading strand of DNA

Answer 20

It can be synthesised continuously.

Question 21

What is the lagging strand? What is it’s key characteristic?

Answer 21

The strand that moves in 5’ to 3’ direction. It is synthesised discontinuously because the DNA polymerase III moves in the same direction.

Question 22

How is the lagging strand synthesised?

Answer 22

Using DNA primase and DNA polymerase III, they produce multiple short sections called Okazaki fragments.

Question 23

State all the steps of DNA replication on the lagging strand.

Answer 23

• DNA primase synthesis short RNA fragments at the replication fork.

• DNA polymerase III extends from the 3’ end of the primer and makes Okazaki fragments.

• DNA polymerase I removes the RNA primers and fills in the gap.

• DNA ligase joins the nicks on the DNA (position represents the end of the RNA primers).

Question 24

Summarise all the events at the replication fork.

Answer 24

• DNA primase synthesises a short RNA primer

• DNA polymerase III adds nucleotides on the 3’ end, to make most of the new DNA.

• The RNA primers are removed and replaced with DNA by DNA polymerase I.

• The gaps between DNA fragments on the lagging strand are joined by DNA ligase.

Question 25

What is the function of the sliding clamp?

Answer 25

It is a protein that helps DNA polymerase stay attached to the DNA template.

Question 26

How fast does DNA gets synthesised in prokaryotes? What is the error rate?

Answer 26

1000 nucleotides per second. Error rate: ~ 10-8, 10-10.

It is slower in eukaryotes.

Question 27

How does DNA polymerase remain accurate while synthesising DNA?

Answer 27

It self-corrects. The proof-reading activity of DNA polymerase enables wrongly incorporated bases to be excised (cut out).

Question 28

Explain the process of DNA polymerase self-correction.

Answer 28

A base that is wrongly incorporated has the 3’ (-OH) group in a slightly different position than of the correct base.
Replication pauses to allow the mistake to be corrected.

DNA polymerase has a 3’-to-5’ exonuclease (removes base) activity that is able to excise the wrongly incorporated base.

Question 29

How does error occur in DNA replication?

Answer 29

• Frameshifts. Extra nucleotides are inserted or omitted (left out).
Can occur if DNA polymerase dissociates (skips) and re-associates (backwards) with the template or if DNA loops out causing strand slippage.

• Substitutions. An incorrect nucleotide is incorporated.

Question 30

Where do errors caused by strand slippage occur the most?

Answer 30

In repetitive regions.

Question 31

Describe the main characteristics of replication of prokaryotic chromosome.

Answer 31

Circular bacterial chromosome has a single origin of replication (ori).
A replication bubble opens up with two replication forks moving away from each other.

Question 32

How long does it take to replicate the entire human genome?

Answer 32

7 hours, polymerisation is ~2 kb/min

Question 33

What is another key structural feature in eukaryotic chromosomes when replicating?

Answer 33

They have multiple origins of replication per chromosome. This makes it quicker to replicate.

Question 34

Where does the replication fork occur?

Answer 34

At point of unwound.