Eukaryotic DNA Polymerases


Deoxyribonucleic acid (DNA) is replicated and repaired by a family of enzymes called DNA polymerases. Eukaryotic cells have a diversity of these enzymes that, while sharing a common biochemical activity, are specialised for particular roles. Three polymerases are required for the replication of the nuclear genome, with Pol α involved in priming and initial synthesis and Pols δ and ϵ involved in bulk DNA replication. These polymerases are dependent on a large number of other proteins which unwind the DNA and perform other functions essential for efficient DNA synthesis. Polymerases are also involved in DNA repair and many repair‐specific enzymes have been identified. Some repair polymerases can refill a gap generated by removal of damaged DNA, or copy a damaged template, allowing DNA synthesis to proceed across a damaged template. Repair polymerases can also have tissue‐specific functions in lymphoid cells, where they contribute to somatic hypermutation of immunoglobulin genes.

Key Concepts:

  • Catalytic function of DNA polymerases.

  • Concepts of ‘proofreading’ and ‘processivity’.

  • Roles of replicative DNA polymerases needed for chromosome replication and organisation at the replication fork.

  • Function of accessory proteins needed for polymerase function in chromosome replication.

  • Different modes of action of specialised polymerases involved in DNA repair.

  • Catalytic mechanism of polymerases.

  • Assays used to detect polymerase function in vitro.

  • Relevance of DNA polymerases to human disease.

Keywords: DNA replication; DNA repair; DNA synthesis; cell cycle; cancer; genome stability; S phase

Figure 1.

(a) Basic mechanism catalysed by DNA polymerases. Polymerisation of nucleotides on the single‐stranded template requires a primer (RNA or DNA) which provides a 3′‐OH group to which the incoming nucleotide is joined and the direction of synthesis is thus 5′ to 3′. Only nucleotides that correctly base pair with the templates strand (according to A•T, G•C rules) are incorporated. One molecule of pyrophosphate (PPi) is produced per nucleotide incorporated. (b) Steps in the polymerisation reaction. The order of the reaction is binding to the template primer, followed by binding of the dNTP. dNTP is cleaved at the α/β bond to give dNMP that is added on to the chain, PPi is released, and the polymerase translocates along to the next 3′ terminus or dissociates. (c) Mechanism of nucleotidyl transferase mechanism of DNA polymerases. Two Mg2+ ions are coordinated in the active site of the polymerase. Mg2+ ion 1 bridges the α‐phosphate group with the 3′OH of the primer, facilitating nucleophilic attack (indicated by arrow). Mg2+ ion 2 ligands the phosphates of the incoming dNTP. The yellow pentagons represent the ribose sugars of the nucleotides.

Figure 2.

A simplified depiction of eukaryotic DNA replication. Mcm helicase is loaded onto DNA at sites bound by the ORC complex in late M/G1 phase. Following activation by cyclin dependent kinase and Dbf4/Cdc7 kinase (DDK), additional proteins bind to the initiation complex and helicase activity is activated. Polymerase ϵ is loaded during initiation and takes over from pol α once leading strand synthesis has occurred.Pol δ is repeatedly loaded on the lagging strand and is again dependent on pol α for priming. Only a subset of factors involved in initiation and elongation are shown.

Figure 3.

Structure of RB69 polymerase. RB69 is a prokaryotic family B polymerase, and is therefore likely to have a structure representative of eukaryotic replicative polymerases (α,δ,ϵ; Franklin et al., ). (a) Polymerisation mode. DNA template‐primer (pink‐orange) first binds to the polymerase thumb domain, followed by binding of a nucleoside triphosphate (blue), complementary to the template base immediately adjacent to the 3′‐OH of the primer. On binding the dNTP, the finger domain (shown here in the open conformation) rotates (black arrow) towards the palm domain to form a closed ternary complex. This facilitates phosphodiester bond formation between primer 3′‐OH and the α‐phosphate of the incoming dNTP. (b) In the exonuclease (proofreading) mode, the thumb tip rotates to partition the DNA to the exonuclease site (shown in yellow), allowing removal of a noncomplementary base. Reproduced from Patel and Loeb ().



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Further Reading

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Yang W (2014) An overview of Y‐family DNA polymerases and a case study of human DNA polymerase η. Biochemistry 53: 2793–2803.

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Cotterill, Sue, and Kearsey, Stephen(Aug 2014) Eukaryotic DNA Polymerases. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001045.pub3]