Eukaryotic DNA Polymerases

Abstract

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 ().

close

References

Acharya N, Yoon JH, Gali H et al. (2008) Roles of PCNA‐binding and ubiquitin‐binding domains in human DNA polymerase eta in translesion DNA synthesis. Proceedings of the National Academy of Sciences USA 105: 17724–17729.

Arana ME, Seki M, Wood RD, Rogozin IB and Kunkel TA (2008) Low‐fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Research 36: 3847–3856.

Arana ME, Takata K, Garcia‐Diaz M, Wood RD and Kunkel TA (2007) A unique error signature for human DNA polymerase nu. DNA Repair 6: 213–223.

Autexier C and Lue NF (2006) The structure and function of telomerase reverse transcriptase. Annual Review of Biochemistry 75: 493–517.

Betous R, Pillaire MJ, Pierini L et al. (2013) DNA polymerase kappa‐dependent DNA synthesis at stalled replication forks is important for CHK1 activation. EMBO Journal 32: 2172–2185.

Braithwaite EK, Prasad R, Shock DD et al. (2005) DNA polymerase lambda mediates a back‐up base excision repair activity in extracts of mouse embryonic fibroblasts. Journal of Biological Chemistry 280: 18469–18475.

Buisson R, Niraj J, Pauty J et al. (2014) Breast cancer proteins PALB2 and BRCA2 stimulate polymerase eta in recombination‐associated DNA synthesis at blocked replication forks. Cell Reports 6: 553–564.

Church DN, Briggs SE, Palles C et al. (2013) DNA polymerase epsilon and delta exonuclease domain mutations in endometrial cancer. Human Molecular Genetics 22: 2820–2828.

Dumstorf CA, Clark AB, Lin Q et al. (2006) Participation of mouse DNA polymerase iota in strand‐biased mutagenic bypass of UV photoproducts and suppression of skin cancer. Proceedings of the National Academy of Sciences USA 103: 18083–18088.

Eddy J, Vallur AC, Varma S et al. (2011) G4 motifs correlate with promoter‐proximal transcriptional pausing in human genes. Nucleic Acids Research 39: 4975–4983.

Franklin MC, Wang J and Steitz TA (2001) Structure of the replicating complex of a pol alpha family DNA polymerase. Cell 105: 657–667.

Gan GN, Wittschieben JP, Wittschieben BO and Wood RD (2008) DNA polymerase zeta (pol zeta) in higher eukaryotes. Cell Research 18: 174–183.

Guo C, Sonoda E, Tang TS et al. (2006) REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo. Molecular Cell 23: 265–271.

Haracska L, Prakash S and Prakash L (2002) Yeast Rev1 protein is a G template‐specific DNA polymerase. Journal of Biological Chemistry 277: 15546–15551.

Haracska L, Unk I, Johnson RE et al. (2001) Roles of yeast DNA polymerases delta and zeta and of Rev1 in the bypass of a basic sites. Genes & Development 15: 945–954.

Haracska L, Yu SL, Johnson RE, Prakash L and Prakash S (2000) Efficient and accurate replication in the presence of 7,8‐dihydro‐8‐oxoguanine by DNA polymerase eta. Nature Genetics 25: 458–461.

Helleday T (2013) PrimPol breaks replication barriers. Nature Structural & Molecular Biology 20: 1348–1350.

Hogg M, Osterman P, Bylund GO et al. (2014) Structural basis for processive DNA synthesis by yeast DNA polymerase varepsilon. Nature Structural & Molecular Biology 21: 49–55.

Johansson E and Dixon N (2013) Replicative DNA polymerases. Cold Spring Harbor Perspectives In Biology 5: a012799.

Johansson E and MacNeill SA (2010) The eukaryotic replicative DNA polymerases take shape. Trends Biochemical Sciences 35: 339–347.

Kang YH, Galal WC, Farina A, Tappin I and Hurwitz J (2012) Properties of the human Cdc45/Mcm2‐7/GINS helicase complex and its action with DNA polymerase epsilon in rolling circle DNA synthesis. Proceedings of the National Academy of Sciences USA 109: 6042–6047.

Kesti T, Flick K, Keranen S, Syvaoja JE and Wittenberg C (1999) DNA polymerase epsilon catalytic domains are dispensable for DNA replication, DNA repair, and cell viability. Molecular Cell 3: 679–685.

Kohzaki M, Nishihara K, Hirota K et al. (2010) DNA polymerases nu and theta are required for efficient immunoglobulin V gene diversification in chicken. Journal of Cell Biology 189: 1117–1127.

Lange SS, Takata K and Wood RD (2011) DNA polymerases and cancer. Nature Reviews Cancer 11: 96–110.

Lange SS, Wittschieben JP and Wood RD (2012) DNA polymerase zeta is required for proliferation of normal mammalian cells. Nucleic Acids Research 40: 4473–4482.

Larrea AA, Lujan SA, Nick McElhinny SA et al. (2010) Genome‐wide model for the normal eukaryotic DNA replication fork. Proceedings of the National Academy Sciences USA 107: 17674–17679.

Lee YS, Gregory MT and Yang W (2014) Human Pol zeta purified with accessory subunits is active in translesion DNA synthesis and complements Pol eta in cisplatin bypass. Proceedings of the National Academy of Sciences USA 111: 2954–2959.

Ma Y, Lu H, Tippin B et al. (2004) A biochemically defined system for mammalian nonhomologous DNA end joining. Molecular Cell 16: 701–713.

Minko IG, Harbut MB, Kozekov ID et al. (2008) Role for DNA polymerase kappa in the processing of N2‐N2‐guanine interstrand cross‐links. Journal of Biological Chemistry 283: 17075–17082.

Muramatsu S, Hirai K, Tak YS, Kamimura Y and Araki H (2010) CDK‐dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon), and GINS in budding yeast. Genes & Development 24: 602–612.

Nick McElhinny SA, Watts BE, Kumar D et al. (2010) Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proceedings of the National Academy of Sciences USA 107: 4949–4954.

Pachlopnik Schmid J, Lemoine R, Nehme N et al. (2012) Polymerase epsilon1 mutation in a human syndrome with facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”). Journal of Experimental Medicine 209: 2323–2330.

Palles C, Cazier JB, Howarth KM et al. (2012) Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nature Genetics 45: 136–144.

Patel PH and Loeb LA (2001) Getting a grip on how DNA polymerases function. Natural Structural Biology 8: 656–659.

Perera RL, Torella R, Klinge S et al. (2013) Mechanism for priming DNA synthesis by yeast DNA Polymerase alpha. eLife 2: e00482.

Prakash S, Johnson RE and Prakash L (2005) Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annual Review of Biochemistry 74: 317–353.

Prasad R, Bebenek K, Hou E et al. (2003) Localization of the deoxyribose phosphate lyase active site in human DNA polymerase iota by controlled proteolysis. Journal of Biological Chemistry 278: 29649–29654.

Pursell ZF and Kunkel TA (2008) DNA polymerase epsilon: a polymerase of unusual size (and complexity). Progress in Nucleic Acid Research and Molecular Biology 82: 101–145.

Reijns MA, Rabe B, Rigby RE et al. (2012) Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development. Cell 149: 1008–1022.

Sale JE, Batters C, Edmunds CE et al. (2008) Timing matters: error‐prone gap filling and translesion synthesis in immunoglobulin gene hypermutation. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 364: 595–603.

Saribasak H and Gearhart PJ (2012) Does DNA repair occur during somatic hypermutation? Seminars in Immunology 24: 287–292.

Sclafani RA and Holzen TM (2007) Cell cycle regulation of DNA replication. Annual Review of Genetics 41: 237–280.

Sharma S and Canman CE (2012) REV1 and DNA polymerase zeta in DNA interstrand crosslink repair. Environmental and Molecular Mutagenesis 53: 725–740.

Singer WD, Osimiri LC and Friedberg EC (2013) Increased dietary cholesterol promotes enhanced mutagenesis in DNA polymerase kappa‐deficient mice. DNA Repair 12: 817–823.

Spiga MG and D'Urso G (2004) Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast. Nucleic Acids Research 32: 4945–4953.

Stancel JN, McDaniel LD, Velasco S et al. (2009) Polk mutant mice have a spontaneous mutator phenotype. DNA Repair 8: 1355–1362.

Steitz TA (1999) DNA polymerases: structural diversity and common mechanisms. Journal of Biological Chemistry 274: 17395–17398.

Stumpf JD and Copeland WC (2011) Mitochondrial DNA replication and disease: insights from DNA polymerase gamma mutations. Cell and Molecular Life Sciences 68: 219–233.

Vaisman A, Frank EG, McDonald JP, Tissier A and Woodgate R (2002) Poliota‐dependent lesion bypass in vitro. Mutation Research 510: 9–22.

Vidal AE and Woodgate R (2009) Insights into the cellular role of enigmatic DNA polymerase iota. DNA Repair 8: 420–423.

Waga S and Stillman B (1998) The DNA replication fork in eukaryotic cells. Annual Review of Biochemistry 67: 721–751.

Weedon MN, Ellard S, Prindle MJ et al. (2013) An in‐frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy. Nature Genetics 45: 947–950.

Williams JS and Kunkel TA (2014) Ribonucleotides in DNA: origins, repair and consequences. DNA Repair 19: 27–37.

Yamanaka K, Minko IG, Takata K et al. (2010) Novel enzymatic function of DNA polymerase nu in translesion DNA synthesis past major groove DNA‐peptide and DNA‐DNA cross‐links. Chemical Research in Toxicology 23: 689–695.

Yousefzadeh MJ and Wood RD (2013) DNA polymerase POLQ and cellular defense against DNA damage. DNA Repair 12: 1–9.

Zegerman P (2013) DNA replication: polymerase epsilon as a non‐catalytic converter of the helicase. Current Biology 23: R273–R276.

Zhang X, Lv L, Chen Q et al. (2013) Mouse DNA polymerase kappa has a functional role in the repair of DNA strand breaks. DNA Repair 12: 377–388.

Zhu W, Ukomadu C, Jha S et al. (2007) Mcm10 and And‐1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. Genes & Development 21: 2288–2299.

Further Reading

Beard WA and Wilson SH (2014) Structure and mechanism of DNA polymerase beta. Biochemistry 53: 2768–2780.

Burgers PM (2009) Polymerase dynamics at the eukaryotic DNA replication fork. Journal of Biological Chemistry 284: 4041–4045.

Cotterill S (ed.) (1998) DNA Replication, A Practical Approach. Oxford: Oxford University Press.

Cotterill S and Kearsey S (2008) DNA replication: a database of information and resources for the eukaryotic DNA replication community. Nucleic Acids Research 37: (database issue), D837–D839.

DePamphilis ML (ed.) (2006) DNA Replication and Human Disease. New York: Cold Spring Harbor Press.

Hubscher U, Maga G and Spadari S (2002) Eukaryotic DNA polymerases. Annual Review of Biochemistry 71: 133–163.

Johnson A and O'Donnell M (2005) Cellular DNA replicases: components and dynamics at the replication fork. Annual Review of Biochemistry 74: 283–315.

Loeb LA and Monnat RJ Jr (2008) DNA polymerases and human disease. Nature Review Genetics 9(8): 594–604.

Maxwell BA, Xu C and Suo Z (2014) Conformational dynamics of a Y‐family DNA polymerase during substrate binding and catalysis as revealed by interdomain Förster resonance energy transfer. Biochemistry 53(11): 1768–1778.

Vengrova S and Dalgaard JZ (eds) (2009) DNA Replication, Methods and Protocols. New York, NY: Humana Press.

Xia S and Konigsberg WH (2014) RB69 DNA polymerase structure, kinetics, and fidelity. Biochemistry 53: 2752–2767.

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

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Cotterill, Sue, and Kearsey, Stephen(Aug 2014) Eukaryotic DNA Polymerases. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001045.pub3]