Lecture 9. The Enzymology Of DNA Replication Flashcards
What does DNA replication consist of?
A leading strand, synthesised continuously and proceeds in the same direction of the replication fork and a lagging strand, synthesised discontinuously and proceeds in the opposite direction of the replication fork
What are Osaka fragments?
Found on the lagging strand, they are a consequence of synthesis of new DNA in one direction only: new DNA is built in the 5’ → 3’ direction
Why is there no 3’ → 5’ synthesis of new DNA?
In 5’ → 3’ synthesis, a high energy bond is cleaved from the incoming nucleotide, providing high energy for polymerisation. In the hypothetical 3’ → 5’ synthesis, no high energy bond can be cleaved, so the reaction cannot proceed
Why was M13 bacteriophage used for DNA replication experiments?
Has single stranded DNA
Replication by ‘rolling circle’
What is rifampicin?
An inhibitor of E. coli RNA polymerase
Does DNase completely destroy Okazaki fragments?
No
Is the primer for an Okazaki fragment DNA or RNA?
RNA
RNA primer synthesis
DNA primate synthesises an RNA primer t initiate DNA synthesis on the lagging strand
Lagging strand synthesis Part 1
- New RNA primers synthesised by DNA primase
- DNA Pol III extends the RNA primer using dNTPs to make Okazaki fragments on the lagging strands
Lagging strand synthesis Part 2
- The old primers are erased by the 5’ → 3’ exonuclease activity of Pol I and are replaced by new DNA
- The gap/nick is sealed by DNA ligaments, joining the Okazaki fragment to the growing chain
- And so on
Leading strand synthesis Part 1
- DNA helicase unwinds the DNA helix, separating the strands
- DNA primase manufactures RNA primer on the leading strand template
- The primed duplex is captured by Pol III
Leading strand synthesis Part 2
- Clamp holder transfers the two halves of the β clamp to Pol III
- New clamp halves maintain the clamp holder in a state of readiness
- Helicase continues to unwind, and Pol III replicates the leading strand continuously
Processivity
A measure of an enzyme’s ability to catalyse consecutive reactions without releasing its substrate
Does Pol III have a high or a low processivity?
Low
Lagging strand synthesis Part 1
- DNA primase manufactures an RNA primer on the lagging strand template
- The primed duplex is captured by Pol III and clamped. This forces the lagging strand template into a loop
