REVIEWS Nature Reviews Genetics | AOP, published online 15 July 2008; doi:10.1038/nrg2345 DNA polymerases and human disease Lawrence A. Loeb*‡§ and Raymond J. Monnat Jr*|| Abstract | The human genome encodes at least 14 DNA-dependent DNA polymerases — a surprisingly large number. These include the more abundant, high-fidelity enzymes that replicate the bulk of genomic DNA, together with eight or more specialized DNA polymerases that have been discovered in the past decade. Although the roles of the newly recognized polymerases are still being defined, one of their crucial functions is to allow synthesis past DNA damage that blocks replication-fork progression. We explore the reasons that might justify the need for so many DNA polymerases, describe their function and mode of regulation, and finally consider links between mutations in DNA polymerases and human disease. non-processive Lagging strand Our knowledge of human DNA polymerases has under- low catalytic efficiency and are . Eight to One of the two DNA strands gone a striking expansion in the past decade. Since ten of these TLS DNA polymerases seem to be present that is synthesized during DNA the discovery in 1957 of an enzyme that catalyses the in most human cells (FIG. 1b; TABLE 1) and are probably replication. The lagging strand accurate replication of DNA, there has been a progres- found in all mammalian cells. Three TLS polymerases is synthesized by Pol δ in short segments that are known as sive accumulation of evidence for five ‘classical’ DNA have been identified in Saccharomyces cerevisiae, and 14 Okasaki fragments. polymerases in all mammalian cells, each functioning two are known in Escherichia coli . in DNA replication and/or repair (FIG. 1a). In an exciting New technologies are helping to establish the roles of Leading strand and unanticipated development that has unfolded over human DNA polymerases in cellular functions. These One of the two DNA strands the past decade, several ‘new’ DNA polymerases have technologies include the creation of cells and animals that is synthesized during DNA 1 (FIG. 1b) replication. The leading strand been discovered . containing mutant DNA polymerases, methods to con- is believed to be synthesized The five classical DNA polymerases are each located trol the expression of specific DNA polymerases, and by Pol ε, predominantly in a on a different chromosome (FIG. 1b). Their principal techniques to quantify the frequency and types of muta- single segment. functions have been deduced from catalytic properties tions in diverse cell types. We are thus well positioned to and observations of cell physiology. DNA polymerase understand the function of each human DNA polymer- (Pol) α catalyses initiation of chromosomal DNA repli- ase in cellular processes, including roles in mutagenesis cation at origins and at Okazaki fragments on the lagging and disease. Phenotypes associated with genetic diseases strand2,3; Pol β participates in base-excision repair4,5; Pol γ can be particularly informative of physiological function; catalyses mitochondrial DNA synthetic processes6; Pol δ however, only a limited number of inherited diseases participates in lagging-strand synthesis7,8; and Pol ε has a have thus far been associated with mutations in DNA *Department of Pathology role in the synthesis of the leading strand of chromosomal polymerases, or with changes in the expression of spe- University of Washington, DNA9–11. Looking back, we can see that the classical list cific DNA polymerases or their associated proteins. This K-072 HSB, BOX 357705, did not take into account the multiplicity and diversity reflects the fact that DNA polymerases were studied most Seattle, Washington DC 98195-7705, USA. of DNA transactions in cells. Each human cell undergoes extensively as enzymes that are essential for DNA replica- 12 ‡Gottstein Memorial Cancer >50,000 DNA-damaging events per day , and much of tion and repair. Each of the classical DNA polymerases Research Laboratory and this damage impedes synthesis by the classical DNA was found to be required for viability, posing a major §Department of polymerases. Given that some lesions are transferred to impediment to assignment of function on the basis Biochemistry and 13 ||Department of Genome progeny cells , there must be mechanisms by which DNA of phenotypes induced by genetic manipulation. Sciences, University of synthesis proceeds past sites of damage. We now know Excellent recent reviews have focused on DNA Washington, Seattle, that synthesis past damage is catalysed by translesion polymerase structure and function15, DNA replica- Washington DC 98195, USA. synthesis (TLS) DNA polymerases (also known as bypass tion16,17, the role of accessory proteins in DNA replication e-mails: DNA polymerases) that can insert nucleotides opposite and repair18,19, and specialized or TLS polymerases20. [email protected]; [email protected] specific blocking lesions, and/or extend the nucleotides Here, we discuss the main unanswered questions con- doi:10.1038/nrg2345 that are inserted opposite damage sites. These enzymes cerning the role of DNA polymerases in maintaining Published online 15 July 2008 are error prone when copying undamaged DNA, have genetic stability; the association of DNA polymerases 594 | AUGUst 2008 | VolUME 9 www.nature.com/reviews/genetics © 2008 Macmillan Publishers Limited. All rights reserved. REVIEWS a Polymerase Gene Catalytic subunit Accessory subunits (kDa) 3′→5′ exonuclease Fidelity Primary function Pol α POLA1 49 58 70 No 10–4–10–5 RNA and/or DNA primers 1462 aa PRIM1 PRIM2A PRIMA2 Pol β POLB None No 5 × 10–4 Base-excision repair 335 aa Pol γ POLG1 55 Yes 10–5 Mitochondrial DNA replication and repair 1239 aa POLG2 Pol δ POLD1 50 68 12 Yes 10–5–10–6 Lagging-strand synthesis DNA repair 1107 aa POLD2 POLD3 POLD4 Pol ε POLE 59 12 17 Yes 10–6–10–7 Leading-strand synthesis 2286 aa POLE2 POLE4 POLE3 b REV7/ MAD2L2 POLN TERT POLS POLE4 POLH POLD2 PRIM2 POLM POLB REV1 POLA2 POLQ POLK POLD4 PRIM1 POLD3 REV3L TDT POLE3 POLL TERC POLE 1 2 3 4 5 6 7 8 9 10 11 12 POLA1 POLI POLD1 POLE2 POLG2 POLG1 13 14 15 16 17 18 19 20 21 22 XY POL families A family B family X family Y family Other Figure 1 | Classical DNA polymerases and associated subunits. a | Five classical DNA polymerasesNatur ande Re theirviews subunits. | Genetics Polymerase catalytic subunits are listed first, and are shown as polypeptides in which catalytic motifs (red lines) and exonuclease motifs (purple lines) are indicated, together with their corresponding genes and number of amino acids. Non-processive These are followed by columns listing the genes for and size of any accessory subunits; whether the holoenzyme has Processivity refers to the proofreading exonuclease activity; and the fidelity of DNA synthesis given as the frequency of single-base substitutions number of nucleotide using gapped M13 DNA as a template. Finally, the primary functions of the polymerases are listed. Data are from Kunkel additions per binding event and co-workers65 or from our unpublished results. b | Location of DNA polymerase genes (POL) in the human genome. The between DNA polymerase locations of 29 genes encoding DNA polymerases and related proteins, including DNA primase genes (PRIM), have been and a DNA template. Non- plotted on a G-banded human karyotype shown at –320 band resolution. The four human DNA polymerase gene and processive DNA polymerases protein families (A, B, X and Y) are colour coded, and were originally defined on the basis of conserved sequence motifs in incorporate one or a few base their catalytic subunits. Accessory subunits of multi-subunit DNA polymerases are shown in the same colour code, and are pairs per DNA-binding event. Processive DNA polymerases typically numbered in order of descending molecular weight after the catalytic subunit. Two other functionally related incorporate thousands of proteins are also shown: genes for the RNA (TERC) and protein (TERT) components of telomerase, and for terminal nucleotides per DNA-binding deoxynucleotidyl transferase (TDT). Designated gene symbols have been used throughout, with the exception of the event. second subunit of DNA polymerase ζ, which is listed as the widely used alias REV7 as well as the official symbol MAD2L2. natURE REViews | GENETICS VolUME 9 | AUGUst 2008 | 595 © 2008 Macmillan Publishers Limited. All rights reserved. REVIEWS Table 1 | Error-prone human DNA polymerases* Polymerase Gene Family Other names Proposed function η (eta) POLH Y RAD30A, XPV Bypass UV lesions ι (iota) POLI Y RAD30B Bypass synthesis κ (kappa) POLK Y DINB1 Bypass synthesis λ (lambda) POLL X POL4 (in Saccharomyces cerevisiae) Base-excision repair, NHEJ µ (mu) POLM X – NHEJ θ (theta) POLQ A Mus308 (in Drosophila melanogaster) DNA repair ζ (zeta) POLZ B REV3 Bypass synthesis Rev 1 REV1 Y REV1L Incorporation of dC opposite abasic sites n (nu) POLN A – Unknown, but Pol n has a unique error signature, G-dTMP mismatches111 *Nomenclature is according to Ref. 110. For family classification of polymerase genes, see fig. 1b. POLQ also encodes a helicase domain of unknown function. Mus308, mutagen sensitive 308; NHEJ, non-homologous end joining; UV, ultraviolet; XPV, xeroderma pigmentosum-variant. with human diseases; the multiplicity, functions and activities remove non-complementary or altered nucle- redundancy of DNA polymerases; regulation of their otides immediately after phosphodiester-bond forma- expression; and the potential of DNA polymerases as tion and before the addition of another nucleotide. The targets for disease prevention and therapy. In addition picture that emerges of DNA polymerases is thus one of to reviewing current knowledge, we discuss areas of constant, repetitive motion with tight coordination research that might have practical importance but for of the polymerase and exonuclease active sites. which we have insufficient data. For example, in the case of cancer, can SNPs in DNA polymerase genes be used Understanding the molecular basis of fidelity.