Lecture 9 - Enzymology of DNA replication

Question 1

Who established the directionality of DNA synthesis?

Answer 1

The Okazaki

Question 2

What is the leading and lagging strand?

Answer 2

Leading: synthesised continuously - same direction as the replication fork

Lagging: synthesised discontinuously - 5’to4’ synthesis proceeds in the opposite direction

Question 3

What are Okazaki fragments?

Answer 3

Small fragments of strand on the lagging strand only

Consequence of synthesis of new DNA in one direction only

Occur away from replication fork

Question 4

Why is there no 3’to5’ synthesis of DNA?

Answer 4

One nucleotide removed leaving 5’ phosphate but a triphosphate is required

No high energy bond can be cleaved, no reaction can be processed

High energy bond required for incorporation of nucleotide

Question 5

What is the life cycle of a M13 bacteriaphage?

Answer 5

Injection via the pilus

Single stranded DNA is replicated and becomes double stranded

Packed into fresh phage and secreted

Question 6

What did Okazaki and Kornberg discovery with the beginning to DNA replication?

Answer 6

DNase cannot completely destroy Okazaki fragments

Primer for an Okazaki fragment is RNA, not DNA

Little pieces of RNA, 10-12 bases long were left

Question 7

How are RNA primers synthesised?

Answer 7

DNA primase is a rifampicin-sensitive DNA directed RNA polymerase

Synthesizes an RNA primer to initiate DNA synthesis on lagging strand

RNA polymerases don’t require a primer

Question 8

How is the lagging strand synthesised?

Answer 8

1. New RNA primers are synthesised by DNA primase
2. DNA Pol III extends RNA primer using dNTPs to make Okazaki fragments on lagging strand
3. As replication fork separates fork separates more DNA, new primers are laid down by DNA primase
4. Old primers erased by 5’to3’ exonuclease
5. Gap sealed with DNA ligase, joining Okazaki fragment to growing chain

Question 9

How is the Okazaki fragment joined by DNA ligase?

Answer 9

DNA ligase uses ATP, releasing pyrophosphate and attaching AMP to 5’ phosphate of downstream fragment

AMP released, phosphodiester bond formed between 3’ -OH of upstream Okazaki fragment and 5’ phosphate of downstream fragment

Sealing needs ATP
New bond seal gap

Question 10

What is the clamp holder?

Answer 10

Hold 2 molecules of Pol III

Has helicase and DNA primase

Question 11

What is leading strand synthesis?

Answer 11

1. DNA helicase unwinds DNA helix, separating strands
2. DNA primase synthesises DNA primer on leading strand template
3. Primed duplex is captured by Pol III
4. New clamp halves maintain in clamp holder

5.Clamp holder transfers 2 halves of B-clamp to Pol III

6. Helicase continues to unwinds, and Pol III replicates the leading strand continuously

Question 12

What is processivity?

Answer 12

Measure of an enzyme’s ability to catalyse consecutive reactions without releasing substrate

Question 13

What is particular of Pol III?

Answer 13

Has low processivity

Can only make short stretches of DNA before it falls off DNA

Question 14

What is the process of lagging strand synthesis?

Answer 14

1. DNA primase produces RNA primer
2. Primed duplex is clamped by Pol III forming loop
3. Helicase continues to unwinds, Pol III extends new primer on lagging strand until Okazaki fragment has been pulled back to Pol II
4. Lagging strand and template are unclamped
5. DNA primase primes the lagging strand template
6. DNA Pol I and DNA ligase repair the gap
7. Process restarts by clamping new lagging strand primer

Question 15

Why does DNA Pol III have to have low processivity?

Answer 15

If it was a highly processive enzyme, it could not release the new Okazaki fragment easily

Question 16

Does DNA Pol I have low or high processivity?

Answer 16

Low

Question 17

When can stem-loop structures be formed?

Answer 17

When DNA is denatured and fails to re-anneal properly

Question 18

What is SSB and what is its importance?

Answer 18

Single stranded DNA binding protein

DNA replication required a supporting cast of SSB

It protects the ssDNA from base pairing and from nuclease

Being displaced by Pol III and replaced as the helix is unwound

Question 19

Why is most DNA negatively supercoiled?

Answer 19

Easier to replicate

Topoisomerases are used to regulate the degree and type of supercoiling

Arises from unwinding of DNA

Question 20

How is overwound positively supercoiled DNA turned into underwound negatively supercoiled DNA using Type II topoisomerase?

Answer 20

1. Topo II binds to the positive supercoil
2. Binding and then cutting BOTH strands of DNA
3. Brings coil forward and re-ligates it causing negative supercoil

Question 21

How is negatively supercoiled DNA relaxed using Type I topoisomerase?

Answer 21

1. Topo I binds to negative supercoil
2. Cuts one DNA strand
3. Causes DNA to unwinds and re-ligates it causing relaxed DNA

Question 22

How is bacterial DNA polymerisation bi-directional?

Answer 22

2x Pol II complexes enter DNA at an origin of replication

Proceeds in both directions at the same time

Topo IV separates the catenated daughter chromosomes by a double stranded break and relegation

Question 23

How quickly do eukaryotic DNA Pols polymerise at?

Answer 23

50 nucleotides per second

Question 24

Why do chromosomes get shorter with each replication?

Answer 24

When DNA polymerase falls off and Okazaki fragments are left so they can join

Primers are erased and gap is filled by DNA polymerase and repaired by DNA ligase on leading strand

Gap on lagging strand cannot be filled by DNA polymerase as there is no primer

Question 25

How is telomerase a reverse transcriptase?

Answer 25

Telomerase provides an RNA template to synthesise a DNA copy of the template at the 3’ end of the parental lagging strand template

Telomeres are build of repetitive motifs

Question 26

Where is telomerase active?

Answer 26

Some germline cells

Epithelial cells

Haematopoietic cells

and in >90% of cancer cell lines
- responsible for immortal phenotype of cancer cells