Helicase

Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs
This occurs at specific regions (origins of replication), creating a replication fork of two strands running in antiparallel directions

DNA Gyrase

DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase
It does this by relaxing positive supercoils (via negative supercoiling) that would otherwise form during the unwinding of DNA

Single-Stranded Binding (SSB) Proteins

SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing
These proteins also help to prevent the single-stranded DNA from being digested by nucleases
SSB proteins will be dislodged from the strand when a new complementary strand is synthesized by DNA polymerase III

DNA Primase

DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template strands
The RNA primer provides an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one

DNA Polymerase III

Free nucleotides align opposite their complementary base partners (A = T ; G = C)
DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
- On the leading strand, DNA pol III is moving towards the replication fork and can synthesize continuously
- On the lagging strand, DNA pol III is moving away from the replication fork and synthesizes in pieces (Okazaki fragments)

DNA Polymerase I

As the lagging strand is synthesized in a series of short fragments, it has multiple RNA primers along its length
DNA polymerase I remove the RNA primers from the lagging strand and replaces them with DNA nucleotides

DNA Ligase

DNA ligase joins the Okazaki fragments together to form a continuous strand
It does this by covalently joining the sugar-phosphate backbones together with a phosphodiester bond