L4 - DNA Replication

Question 1

What direction does DNA replication occur?

Answer 1

5’ to 3’ direction by formation of phosphodiester bonds
Irreversible reaction because coupled to breakdown of PPI to 2PI by pyrophosphatase
Template and primer strand are anti-parallel

Question 2

Is DNA replication exothermic or endothermic?

Answer 2

Exothermic

Free energy provided by the breakage of two high energy phosphate bonds

Question 3

DNA replication steps

Answer 3

1. Replication fork
2. DNA helicase separates paired strands using ATP
3. Extension of RNA primer by DNA primase - NTPs
4. New strands cannot both be synthesised continuously
   - Lagging strand - discontinuous Okazaki fragments (right to left)
   - Leading strand - continuous (left to right)
5. DNA ligase uses energy from ATP hydrolysis to ligate newly synthesised DNA fragments

Question 4

Lagging strand synthesis requires

Answer 4

DNA primase - makes RNA primer 
DNA polymerase - extends the primer 
RIbonuclease H - removes RNA primer 
DNA polymerase - extends across the gap 
DNA ligase - seals the nick

Question 5

Mutations in genes encoding DNA helicases cause which diseases?

Answer 5

Werner syndrome - premature ageing
- Mutations are recessive in RECQ helicase WRN gene
Bloom syndrome - rare cancer syndrome
- Loss of function mutation in RECQ DNA helicase
- Loss of genome integrity

Question 6

Processivity of DNA polymerases

Answer 6

Processivity of DNA polymerase is enhanced association with a sliding clamp
Once first step of DNA synthesis has finished, interaction of DNA polymerase with primer:template junction is maintained and addition of nucleotides is rapid.
Sliding clamp is positioned near junction by a clamp loader using energy from ATP hydrolysis
ATP dependent
Sliding clamps help move he DNA polymerase forward

Question 7

Single stranded binding proteins function

Answer 7

Expose single-stranded DNA in the replication fork so it can be used for synthesis
Keep replication fork open
Enhance processivity of DNA polymerase
Once bound the probability of another one binding I higher - straightens chain

Question 8

DNA topoisomerases function

Answer 8

Prevent DNA tangling during DNA replication
Nick and reseal the backbone of the parental helix
- Unwinding of DNA strands at replication fork introduce super helical

Question 9

Two types of DNA topoisomerases

Answer 9

1 - nick and reseal 1 of 2 strands - no ATP

2 - nick and reseal both strands - ATP

Question 10

Control of DNA replication

Answer 10

DNA replicators and origins direct initiation of DNA replication by recruiting replication initiator proteins

Question 11

Control of DNA replication - Yeast

Answer 11

Autonomously replicating sequence elements

Question 12

Control of DNA replication - Humans

Answer 12

DNA sequences near to LMNB2, MYS and HBB

Question 13

Initiation of DNA replication in eukaryotes

Answer 13

Biphasic

• Replicator selection - formation of pre-replicative complex - G1
• Origin activation - unwinding of DNA and recruitment of DNA polymerase - S

Temporal selection of these two events - ensures each origin is used and each chromosome is only replicated once per cycle

Question 14

Replicator selection protocol

Answer 14

1. Origin recognition complex binds to replicator sequence
2. Helicase loading proteins bind to origin recognition complex
   - cdc6 and cdt1
3. Helicase Mcm2-7 binds to complete formation of pre-replicative complex
   High levels of CDK activity in S phase activate existing pre-replicative complex

Question 15

Problem with RNA primer

Answer 15

Created an end replication problem for linear eukaryotic chromosomes

Question 16

Incompletely replicated DNA

Answer 16

DNA polymerase and ligase close all but one gap
Incomplete replicated DNA so end sequence is lost
- DNA molecule with telomere
- Ribonuclease H removes RNA primer further shortening strand

Question 17

Compensating for loss of telomere sequence

Answer 17

Addition of TTAGGG repeats by telomerase
Extended 3’ end is long enough to enable DNA primase to bind and initiate RNA primer synthesis
- Extended as an extra Okazaki fragment

Question 18

Telomerase

Answer 18

Contains an RNA component that specified telomere sequence
Ribonucleoprotein with an intrinsic RNA component
Acts as a template on which telomere repeat sequences are synthesises - telomerase shuffle
Telomerase RNA allows addition of multiple TTAGGG repeats to the 3’ OH end

Question 19

Key components of the E. coli and human Replication Forks are?

Answer 19

Highly conserved