9 - Enzymology of DNA Replication

Question 1

Direction of DNA synthesis in each anti-parallel strand

Answer 1

leading - 5’ to 3’
lagging - 3’ to 5’

Question 2

summary of DNA replication so far

Answer 2

• DNA replication is semi-conservative
• DNA strands are anti-parallel
• DNA polymerase exonuclease enzyme used to form phosphodiester bonds between adjacent nucleotides
• complemntary base pairs joined by Watson-Crick pairing - weak hydrogen bonds
• DNA synthesis is semi-continuous, with a leading and lagging strand
  leading - 5’ to 3’
  lagging - 3’ to 5’

Question 3

1968 - Okazakis research

Answer 3

-cestablished directionality (polarity) of DNA synthesis in replication
-cleading strand synthesised continuosly in same direction of replication fork (5’ to 3’)
-clagging strand synthesised discontinuosly in opposite direction to that of replication fork (3’ to 5’)
- theorised okazaki fragement formation in lagging strand formation - they vary in nucleotide strand size (100-200 to 1000-2000)

Question 4

what are okazaki fragments

Answer 4

consequence of synthesis of new DNA in one direction only - 5’ to 3’ direction

Question 5

which direction is new DNA built in DNA replication

Answer 5

5’ to 3’ in leading strand

Question 6

why is there no synthesis of new DNA in 3’ to 5’ direction

Answer 6

in 5’ to 3’ in leading strand, editing and proofreading of nucleotide chain is permitted
- editing not permitted in 3’ to 5’ in lagging strand

Question 7

Findings from Okazaki and Kornberg in 1971

Answer 7

Kornberg - replication of M13 phage DNA from single-stranded infective form to double-stranded replicative form by an E. coli extract is prevented by rifampicin
- Rimpaficin is an inhibitor of E. coli RNA polymerase

Okazaki - found that DNAse cannot completely destroy okazaki fragments
- instead left little pieces of RNA, 10-12 bases long

Question 8

what is the primer for an okazaki fragment

Answer 8

RNA not DNA

Question 9

requirements for synthesis of new DNA strand (DNA replication) in all DNA polymerases

Answer 9

• a single stranded DNA template
• a DNA primer base paired with the template
• primer must have a free hydroxyl group at 3’ end of the primer to accept a new nucleotide

-a source of dNTP precursors

Question 10

where energy for polymerisation of nucleotide chain comes from

Answer 10

release of diphosphate via cleavage of two phosphates when adjacent nucleotides join and form a phosphodiester bond.

Question 11

why does DNA replication have very low error rates

Answer 11

proofreading by the DNA polymerases
- depends on the 3’ to 5’ exonuclease activity of some DNA polymerases
- most DNA polymerases have a very high fidelity

Question 12

example of proofreading in some DNA polymerases

Answer 12

e.g. if a base on a nucleotide is the wrong one (not complementary)
- base pairing on the 3’ nucleotide of nascent and template strand does not occur
- polymerase enzyme then pauses and removes the incorrect mispaired base
- then transfers the 3’ end back to polymerase binding site
- where this region is copied and joined correctly

Question 13

DNA replication fork cooperation - general mechanism (textbook)

Answer 13

• nucleotides added by a DNA polymerase to each growing daughter strand in the 5’ to 3’ direction
• leading strand synthesised continuously from a single RNA primer at its 5’ end
• lagging strand synthesised discontinuously from multiple RNA primers that are formed periodically as each new region of of the parent duplex is unwound
• elongation of these RNA primers produces okazaki fragments
• as each growing okazaki fragment reahces the previous primer,
• primer is removed and the fragments are ligated (by DNA ligase)
• repetition results in the synthesis of the entire lagging strand

Question 14

model used for DNA replication

Answer 14

SV40 DNA replication machine
- SV40 is a single-stranded virus

Question 15

M13 life cycle

Question 16

RNA primer synthesis and info

Answer 16

• DNA primase is a rifampicin-sensitive DNA-directed RNA polymerase
• DNA primase synthesis and RNA primer to initiate DNA synthesis on the lagging strand
• RNA polymerase do not need a primer

Question 17

lagging strand synthesis mechanism (Which one to use???)

Answer 17

• ??? - look at two different mechanisms on slides

Question 18

Okazaki fragment joining by DNA ligase

Answer 18

• DNA ligase uses ATP as an energy source
• releases pyrophosphate and attaches AMP to the 5’ phosphate of the downstream fragment
• AMP is released and a phsophodiester bond is formed between the 3’-OH of the upstream Okazaki fragment and the 5’ phosphate of the downstream fragment

Question 19

leading strand synthesis mechanism

Answer 19

• DNA helicase unwinds the DNA helix, separates the strands
• DNA primase manufactures an RNA primer on the leading strand template
• the primed duplex is captured by Pol III
• Pol III has a low processivity - it can only make short stretches of DNA before it falls of the DNA
• new clamp halves maintain the clamp holder in a state of readiness
• the clamp holder transfers the two halves of the beta-clamp to Pol III
• clamping converts Pol III to hig processivity - it can now replicate long stretches of DNA
• Helicase continues to unwind
• Pol III replicates the leading strand continuously

Question 20

processivity def (enzymes)

Answer 20

a measure of an enzyme’s ability to catalyse consecutive reactions without releasing its substrate.