DNA REPLICATION

Question 1

DNA replication is ……………- ………………………

Answer 1

Semi Conservative

Question 2

In what direction does DNA replication occur?

Answer 2

5’ to 3’

Question 3

How does a phosphodiester bond form?

Answer 3

Incoming dNTP undergoes nucleophilic attack by the 3’OH on the P of dNTP.

Question 4

If DNA are anti-parallel to each other, what issue does this create?

Answer 4

DNA polymerase can only act in the 5’ to 3’ direction and so the leading strand can only be synthesised in this manner.

Question 5

What regulated replication?

Answer 5

The replisome.

Question 6

How is the lagging strand synthesised?

Answer 6

Lagging strand synthesis requires DNA primase, DNA polymerase, Ribonuclease H, and DNA ligase to convert Okazaki fragments into a continuous strand.

Question 7

What makes the RNA primer?

Answer 7

DNA primase.

Question 8

How does lagging strand synthesis occur?

Answer 8

DNA helicase unwinds the helix
DNA primase makes the RNA primer on the single strand
DNA polymerase can extend the primer and add nucleotides.
For the laggings strand several primers are needed for templating in the 5’ - 3’.
The primers need to be removed by Ribonuclease H which specifically degrades RNA on an RNA DNA hybrid molecule. The gap left is filled by DNA polymerase bu there is still a small nick in the DNA. This must be sealed by DNA ligase.
Hence from Okazaki fragments you can get a continuous strand.

Question 9

How does DNA Helicase work?

Answer 9

Uses ATP to separate the parental DNA strands (H bonds) at the replication fork and moved the replication forwards.

Question 10

Mutations encoding for DNA helicases causes diseases such as what?

Answer 10

Werner’s Syndrome, a proeria caused by an autosomal recessive mutation in the RECQ helicase gene WRN.
Evidence that the mutant helicase is shortened, not transported to the nucleus or broken down too quickly.

Question 11

What kind of cancer can be caused by mutations in DNA Helicases?

Answer 11

Bloom Syndrome: a cancer syndrome formed by LOF in the RECQ family DNA helicase which maintains genome integrity.
BLM gene not expressed.

Question 12

What is processivity?

Answer 12

enzyme ability to catalyse consecutive reactions without releasing its substrate.

Question 13

What enhances processivity of DNA polymerase?

Answer 13

1. Sliding clamp association
2. Single stranded DNA binding proteins
3. Topoisomerases

Question 14

What is the sliding clamp?

Answer 14

Once DNA primase has made an RNA primer, two factors complete replisome assembly: the clamp loader and sliding clamp. The sliding clamp and clamp loader form in an ATP dependent manner. This complex triggers recruitment of DNA polymerase.

Question 15

What are single stranded binding proteins?

Answer 15

Another protein that adds processivity to DNA polymerase. Binding proteins stop the the single stranded DNA binding to itself so that the replication fork does not stall.

Question 16

What are DNA Topoisomerases?

Answer 16

Enhance processivity of DNA polymerase by preventing DNA from becoming tangled in replication.

Question 17

How do DNA Topoisomerases work?

Answer 17

Type 1 - nick and reseal one strand of DNA passively to release tension.
Type 2 - nick and reseal both strand of DNA to release tension but by use of ATP.

Question 18

What is an origin of replication?

Answer 18

A specific DNA sequence where replication starts. These specific DNA sequences recruit replication initiator proteins.

Question 19

What are the origins of replication in Yeast?

Answer 19

Approximately 600-700 Autonomously Replicating Sequences

Question 20

What are the origins of replication in E Coli?

Answer 20

Replication begins at a single genetically define replicator site called oriC.

Question 21

Origins of replication in humans is poorly understood. What do we know?

Answer 21

100,000+ origins

LMNB2 and MYC seem to be involved.

Question 22

Initiation of DNA replication from the origin of replication is biphasic. What are these phases?

Answer 22

1. Replicator Selection (G1) - formation of a pre replicative complex
2. Origin Activation (S phase) - unwinding of DNA and recruitment of DNA polymerase

Question 23

What is the initiation of DNA replication in eukaryotes biphaic and separated temporally?

Answer 23

To preserve integrity of the genome, each origin of replication must fire no more that once per cell cycle.

Question 24

What happens in The Replicator Selection that occurs in G1 (the first part of replication initiation)?

Answer 24

The Replicator Selection phase.
An origin recognition complex binds to a replicator sequence to which helicase loading proteins and helicase binds.
This is the complete formation of the Pre replicative complex in G1.

Question 25

What happens in the Origin Activation phase in S phase (the second part of replication initiation?)

Answer 25

High levels of Cyclin dependent kinase activity activates the existing pre replicative complex and inhibits formation of new pre replicative complexes.

Question 26

Why is there a close relationship between pre replicative complex function, CDK level and the cell cycle?

Answer 26

To ensure that chromosomes are replicated exactly once per cycle.

Question 27

What is the problem encountered at the end of DNA replication?

Answer 27

DNA polymerase cannot replicate DNA de novo; they can only extend from an RNA primer that sits on the very end of a DNA molecule. When said primer is removed by Ribonuclease H, there is no upstream DNA for a primer to bind and no DNA polymerase that can function independently and so there is an overhang on the 3’ of single stranded DNA that cannot be copied at 5’ end on chromosomes.

Question 28

What does an overhang at the end of replication have implications on and how is this resolved?

Answer 28

The overhang means there is loss of valuable coding information and the chromosome risks being shortened. To prevent this telomerase adds TTAGGG repeats to the 3’ end of every replicated molecule of DNA. It adds enough repeats that a new RNA primer can be created and a new fragments can be made so that no coding information is lost.

Question 29

How does telomerase work?

Answer 29

Telomerase is a ribonucleoprotein that contains an RNA and protein component that acts as a template for temolere sequences to be synthesised. This occurs in a stepwise process called the Telomerase shuffle, allowing movement 6 nucleotides at a time.