BIOCHEMISTRY

DNA replication in bacteria

• coli DNA polymerase I requires all four deoxynucleoside 5′-triphosphates (dNTPs) as precursors, Mg2_, a DNA template and a primer with a 3’-OH end. DNA synthesis occurs in a 5’ → 3’ direction.
• DNA polymerase I also has a 3’ → 5’ exonuclease (proof-reading) activity and a 5’ → 3’ exonuclease activity. coli DNA polymerases II and III lack the 5’ → 3’ exonuclease activity.

• Replication starts at a single origin, is bi-directional and semiconservative.

• Each replication bubble (or eye) consists of two replication forks
• DNA synthesis proceeds in a 5’ → 3’ direction on each strand of the parental DNA. On the strand with 3’ → 5’ orientation (the leading strand) the new DNA is synthesized continuously. On the strand that has 5’ → 3’ orientation (the lagging strand) the DNA is synthesized discontinuously as a series of short Okazaki fragments that are then joined together

• DNA replication requires an RNA primer that is synthesized by an RNA polymerase called primase. This is extended by DNA polymerase III, which makes the DNA for both the leading and lagging strands. DNA polymerase degrades the primer and replaces it with DNA. DNA ligase then joins DNA ends.

A helicase unwinds the DNA double helix and single-stranded DNAbinding (SSB) protein stabilizes the single-stranded regions during replication. DNA topoisomerase I is needed to allow the helix to unwind without causing extensive rotation of the chromosome. DNA topoisomerase II separates the two daughter DNA circles following replication