Replisome

A representation of the structures of the replisome during DNA replication

The replisome is a complex molecular machine that carries out replication of DNA. It is made up of a number of subcomponents that each provide a specific function during the process of replication.

Major components

While the replisome acts as a singular machine, its subcomponents are the complexes that perform the specific functions of replication.

Helicase

Helicase is an enzyme which breaks H-bonds between the two strands of DNA. Its doughnut like structure wraps around DNA and separates the strands ahead of DNA synthesis. As helicase unwinds the double helix, it causes the helix to form supercoils in other areas of the DNA. In eukaryotes, the Mcm2-7 complex acts as a helicase, though which subunits are required for helicase activity is not entirely clear. This helicase translocates in the same direction as the DNA polymerase (3' to 5' with respect to the template strand). In prokaryotic systems, the helicases are better identified and include dnaB, which moves 5' to 3' on the strand opposite the DNA polymerase.

Gyrase

Gyrase (a form of Topoisomerase) relaxes and undoes the supercoiling caused by helicase. It does this by cutting the DNA strands, allowing it to rotate and release the supercoil, and then rejoining the strands. Gyrase is most commonly found upstream of the replication fork, where the supercoils form.

Primase

Primase adds complementary RNA primers to a DNA strand to begin Okazaki fragments. These primers are approximately 10 base pairs long and are at the 5' end of the okazaki fragment. Additionally, because DNA Polymerase can continue, but not begin, a strand, Primase must begin the leading strand as well.

DNA polymerase III holoenzyme

DNA polymerase III (DNA pol III) holoenzyme contains two catalytic cores - one for replication of the leading strand and one for the lagging strand. DNA pol III stays on the strands via a dimer beta clamp. There is a bridge between the two catalytic cores. DNA pol III also removes the single-strand binding proteins (see below).

A fully assembled rendering of the PCNA clamp

DNA pol III, on its own, cannot stay on the DNA strand long enough to efficiently replicate a daughter strand. The replisome contains a mechanical-like structure called the beta clamp, which contains three subunits that come together to enclose the strand, which helps DNA pol III stay on the strand for 1000-2000 nucleotides, as opposed to 100-200.

DNA pol III has two key limitations:

• It can only add nucleotides to the 3' end of a strand.
• It cannot start a new strand, it can only extend an existing strand (because it must only add to 3' ends of strand).

These limitations directly influence the requirements of the DNA pol III enzyme to both replicated in a leading/lagging fashion, as well as require primers to begin strands.

DNA polymerase I

DNA polymerase I (DNA pol I) removes the RNA primers set by Primase through exonuclease activity, and (mostly; see ligase below) completes the Okazaki fragments through polymerase activity.

Ligase

Because there is a small gap remaining after pol I continues the strand of the Okazaki fragment, ligase is required to fill in that gap. The two ends of the Okazaki fragments are connected with covalent bonds.

Single-strand binding proteins

Exposed, single-strand DNA is highly unstable (particularly in the aqueous environment of the cell). The single-strand DNA can hydrogen bond to itself and form dangerous hairpin structures. To counteract this instability, single-strand binding proteins (SSB) (e.g. Replication protein A) bind to the exposed bases.

Exonuclease activity

Incorrect base pairing occurs rarely during replication. Some DNA polymerases contain "proofreading" mechanism that removes nucleotides that have been mistakenly added. This is termed as exonuclease activity.

External links

• MeSH DNA+replisome

References

Pomerantz, R. T.; o’Donnell, M. (2007). "Replisome mechanics: Insights into a twin DNA polymerase machine". Trends in Microbiology 15 (4): 156–164. doi:10.1016/j.tim.2007.02.007. PMID 17350265.