DNA polymerase III holoenzyme

Components

The replisome is composed of the following:

• 2 DNA Pol III enzymes, each comprising α, ε and θ subunits. (It has been proven that there is a third copy of Pol III at the replisome.)
  • the α subunit (encoded by the dnaE gene) has the polymerase activity.
  • the ε subunit (dnaQ) has 3'→5' exonuclease activity.
  • the θ subunit (holE) stimulates the ε subunit's proofreading.
• 2 β units (dnaN) which act as sliding DNA clamps, they keep the polymerase bound to the DNA.
• 2 τ units (dnaX) which act to dimerize two of the core enzymes (α, ε, and θ subunits).
• 1 γ unit (also dnaX) which acts as a clamp loader for the lagging strand Okazaki fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits which include 3 γ subunits, 1 δ subunit (holA), and 1 δ' subunit (holB). The δ is involved in copying of the lagging strand.
• Χ (holC) and Ψ (holD) which form a 1:1 complex and bind to γ or τ. X can also mediate the switch from RNA primer to DNA.

Activity

DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second. DNA Pol III activity begins after strand separation at the origin of replication. Because DNA synthesis cannot start de novo, an RNA primer, complementary to part of the single-stranded DNA, is synthesized by primase (an RNA polymerase):

("!" for RNA, '"$" for DNA, "*" for polymerase)

! ! ! ! _ _ _ _ _ _ _ _ | RNA |
G U A U | Pol |

• • • • |_ _ _ _|
        C A T A G C A T C C

$ $ $ $ $ $ $ $ $ $

Addition onto 3'OH

As replication progresses and the replisome moves forward, DNA polymerase III arrives at the RNA primer and begins replicating the DNA, adding onto the 3'OH of the primer:

! ! ! ! _ _ _ _ _ _ _ _ | DNA |
G U A U | Pol |

• • • • |III _|
        C A T A G C A T C C

$ $ $ $ $ $ $ $ $ $

Synthesis of DNA

DNA polymerase III will then synthesize a continuous or discontinuous strand of DNA, depending if this is occurring on the leading or lagging strand (Okazaki fragment) of the DNA. DNA polymerase III has a high processivity and therefore, synthesizes DNA very quickly. This high processivity is due in part to the β-clamps that "hold" onto the DNA strands.

! ! ! ! $ $ $ $ $ $ _ _ _ _ _ _ _ _ _ _ _ _ _ _| DNA |
G U A U C G T A G G| Pol |

• • • • • • • • • *|III _|
                  C A T A G C A T C C

$ $ $ $ $ $ $ $ $ $

Removal of primer

After replication of the desired region, the RNA primer is removed by DNA polymerase I via the process of nick translation. The removal of the RNA primer allows DNA ligase to ligate the DNA-DNA nick between the new fragment and the previous strand. DNA polymerase I & III, along with many other enzymes are all required for the high fidelity, high-processivity of DNA replication.

References

References

1. (2010). "Stoichiometry and Architecture of Active DNA Replication Machinery in Escherichia Coli". Science.
2. (December 1995). "DnaX complex of Escherichia coli DNA polymerase III holoenzyme. The chi psi complex functions by increasing the affinity of tau and gamma for delta.delta' to a physiologically relevant range". J. Biol. Chem..
3. (1995). "DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine". Annu. Rev. Biochem..