DNA polymerase III holoenzyme


DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication. It was discovered by Thomas Kornberg and Malcolm Gefter in 1970. The complex has high processivity and, specifically referring to the replication of the E.coli genome, works in conjunction with four other DNA polymerases. Being the primary holoenzyme involved in replication activity, the DNA Pol III holoenzyme also has proofreading capabilities that corrects replication mistakes by means of exonuclease activity reading 3'→5' and synthesizing 5'→3'. DNA Pol III is a component of the replisome, which is located at the replication fork.

Components

The replisome is composed of the following:
  • 2 DNA Pol III enzymes, each comprising α, ε and θ subunits.
  • *the α subunit has the polymerase activity.
  • *the ε subunit has 3'→5' exonuclease activity.
  • *the θ subunit stimulates the ε subunit's proofreading.
  • 2 β units which act as sliding DNA clamps, they keep the polymerase bound to the DNA.
  • 2 τ units which act to dimerize two of the core enzymes.
  • 1 γ unit 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, and 1 δ' subunit. The δ is involved in copying of the lagging strand.
  • Χ and Ψ 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 :
-------->
* * * *
! ! ! ! _ _ _ _
_ _ _ _ | RNA | <--ribose -phosphate backbone
G U A U | Pol | <--RNA primer
* * * * |_ _ _ _| <--hydrogen bonding
C A T A G C A T C C <--template ssDNA
_ _ _ _ _ _ _ _ _ _ <--deoxyribose -phosphate backbone
$ $ $ $ $ $ $ $ $ $

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 | <--deoxyribose -phosphate backbone
G U A U | Pol | <--RNA primer
* * * * |_III_ _| <--hydrogen bonding
C A T A G C A T C C <--template ssDNA
_ _ _ _ _ _ _ _ _ _ <--deoxyribose -phosphate backbone
$ $ $ $ $ $ $ $ $ $

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 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 | <--deoxyribose -phosphate backbone
G U A U C G T A G G| Pol | <--RNA primer
* * * * * * * * * *|_III_ _| <--hydrogen bonding
C A T A G C A T C C <--template ssDNA
_ _ _ _ _ _ _ _ _ _ <--deoxyribose -phosphate backbone
$ $ $ $ $ $ $ $ $ $

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.