DNA replication

what was Meselson & Stahl’s experiment?

• semi conservative replication.

  • bacteria grown in N15 - all DNA is heavy

  • transferred to N14 medium

  • continued growth in N14 medium

  • formed semi conservative strands

what is the direction of replication?

• bidirectional

• 2 replication forks/origin

where does replication begin in prokaryotes?

• origin of replication is called OriC in prokaryotes

• only one origin of replication in pro., multiple origins in eukaryotes

• DNA replicated from one origin of replication = replicon

• only one replicon in prokaryotes

• Ter regions for termination of replication

where does replication begin in eukaryotes?

• multiple replicons in eukaryotes DNA replicated

• origins 3 and 5 fire first, followed by 1

• origins 2 and 4 are replicated by the elongation of replication forms from origins 3 and 5

• origins 2 and 4 are passively replicated

what is the replicon model of replication initiation?

SV40

• four pentameric sites (P) for initiator protein large T antigen

• early palindrome (EP) sites for DNA unwinding

S.cerevisae:

• A and B1 are recognised by ORC

• B2 is site for DNA unwinding

• red site indicates site of initial DNA synthesis - this is beyond the sequence shown for OriC

what are the stages of replication & initiator proteins?

• oriC (E-coli)

• DNA melting + initiator protein (DnaA)

• recruitment of DNA helicase (DnaB) + loading of helicase onto DNA (DnaC)

• DNA unwinds (ATP → ADP + Pi)

what are the roles of DnaA, DnaB and DnaC?

• DnaA binds to double stranded 9mer regions

• when DnaA is bound to ATP, it can interact with DNA in 13mer regions that results in it separating to single stranded DNA

• DnaA recruit complex DnaB and DnaC, helicase and helicase loader respectively

  • Helicase is inactive when bound by the helicase loader

what is the role of DNA polymerase?

recruited & replication is able to proceed.

what direction does DNA synthesis proceed in?

• in 5’ to 3’ direction

what drugs target DNA synthesis?

• many anti-cancer drugs target DNA synthesis, cancer cells are susceptible since they are rapidly dividing.

• approaches include:

  • diminishing the supply of correctly structured nucleotides

  • including cross linking between adjacent nucleotides which obstructs replication machinery

  • inhibiting the action of topoisomerases

describe lagging strand synthesis

• DNA polymerase III - extends the leading strand

• lagging strand synthesis involves Okazaki fragments

• in prokaryotes, DNA polymerase I removes the RNA primers and extends the DNA

• in eukaryotes, the lagging strand synthesis is carried out by DNA polymerase a and DNA polymerase ?

• ligase seals the gap

how are eukaryotes proteins replicated?

• replication proteins function independently

• leading strands synthesis is carried out by DNA polymerase 3

• lagging strand synthesis is started by DNA polymerase a and extended by DNA polymerase ?

explain termination in circular and linear chromosomes

• catenanes-2 circular linked pieces of DNA

• topoisomerase nicks one DNA double strand and passes the other through the break, separating the two DNA circles

• topisomerase II releases new strand-decatenation

how accurate is DNA replication?

• 10^-9 to 10^-10 per base pair per round of replication

• this is due to:

  • choice of correct base to be inserted (base pairing)

  • proofreading by the DNA polymerase

  • post-replication repair systems

why must errors be fixed?

• first round replication - mis-incorporation (repair)

• second round replication

what are the steps of PCR?

1. denaturation

2. annealing

3. extension

explain the full steps of DNA replication.

• starts at ORC in humans, OriC in E-coli

• local separation of duplex DNA

• helicase unwinds the DNA at the fork

• SSB proteins keep the duplex strands apart

• DNA Primase synthesises a short RNA primer

• DNA polymerases synthesise the new daughter strand in the 5’-3’ direction

• ligase completes the last phosphodiester bond to secure the complete new DNA strand

• topoisomerase ensures correct helical density

what are some important things to remember?

• DNA polymerases can’t melt or unwind duplex DNA

• DNA polymerases can’t initiate chains directly, but can only extend a pre-existing DNA or RNA strand (need primers)

• DNA replication can only start at an origin of replication

• all DNA strands grow in a 5’ to 3’ direction