ACBK001-Ed-Book March 18, 2005 14:4

The Functions and Fidelity of Human

DNA Polymerases
Thomas A. Kunkel
Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina

Background
The ability to synthesize DNA correctly is critical for avoiding mutations that can initiate and promote cancer.
High fidelity replication requires an amazing diversity of DNA transactions. These include several distinct DNA
repair pathways to remove the many types of DNA damage that can result from normal cellular metabolism and
external environmental stresses (1). Most of these repair processes require DNA synthesis by polymerases, and
they ultimately produce undamaged templates for replication. Replication is normally accurate due to the high
nucleotide selectivity of the major replicative DNA polymerases and the ability of their 3
-exonucleases to excise
errors (2). Occasional failure to repair lesions prior to replication can stall the replication machinery, leading to
recruitment of proteins that alleviate the block through fork regression, recombination, or translesion synthesis
(TLS) by specialized DNA polymerases (3). Rare replication errors that are not proofread result in mismatches
can be corrected by the DNA mismatch repair system (MMR) (4). MMR also requires DNA synthesis, and
defects in MMR lead to elevated rates of point mutations and altered responses to DNA damage, ultimately
increasing susceptibility to cancer (5, 6).
To perform the DNA transactions needed for genome stability, the human genome encodes numerous DNA
polymerases classified by homology into different families, A, B, X, and Y (Table 1) (7). More than half of these
have been discovered in the past six years and their cellular functions and biochemical characteristics are the subject
of very active investigation. Eukaryotic DNA polymerases differ widely in mass, structure, associated subunits,
enzymatic activities, and biochemical properties, especially including the fidelity with which they synthesize
DNA. Briefly considered here are their fidelity in relation to normal functions and two strong associations
between altered DNA polymerase function and cancer (8).

Discussion
The most common mistakes made by DNA polymerases are single base substitution and single-base deletion
errors. The error rates for nine different template-dependent mammalian DNA polymerases are listed in Table 2.
These are average error rates for synthesis in vitro to fill a single stranded gap of several hundred nucleotides.
Consistent with the need for high chromosomal replication fidelity in vivo, the major replicative polymerases
(Pol ε, Pol δ, and mitochondrial Pol γ ) that have intrinsic proofreading exonucleases are the most accurate.
Comparing these error rates to those for their proofreading-defective derivatives (not shown) reveals that high
fidelity typically results from 104 to 106 –fold selectivity for inserting correct rather than incorrect nucleotides,
followed by proofreading of 90% to 99.9% of mismatches. The potential relevance of proofreading of replication
errors to human cancer is implied by the strong increase in several types of cancer in mice in which the proofreading
exonuclease of Pol δ has been inactivated (9).
These polymerases have other functions likely to be relevant to cancer. Pol ε functions in cell cycle checkpoint
control in response to DNA damage, and Pol δ and Pol ε are also thought to fill DNA gaps during mismatch repair,

© 2005 American Association for Cancer Research.

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The Functions and Fidelity of Human DNA Polymerases

Table 1. Human DNA polymerases

Polymerase Family Human gene Mol. Wt. (kDa) 3
Exo Other activities

γ (gamma) A POLG 140 Yes dRPlyase

θ (theta) A POLQ 290 No ATPase, helicase
ν(nu) A POLN 100 No
α(alpha) B POLA 165 No RNA primase
δ(delta) B POLD1 125 Yes
ε(epsilon) B POLE 225 Yes
ζ (zeta) B POLZ (REV3) 353 No
β(beta) X POLB 39 No dRP & AP lyase
λ(lambda) X POLL 66 No dRP lyase, TdT
µ(mu) X POLM 55 No TdT
TdT X 56 No
σ (sigma) X POLS (TRF4-1) 60 No
η(eta) Y POLH (RAD30A, XPV) 78 No
ι(iota) Y POLI (RAD30B) 80 No dRP lyase
κ(kappa) Y POLK (DINB) 76 No
Rev1 Y REV1 138 No

nucleotide excision repair, and long-patch base excision repair. Although error rates for DNA repair reactions
in vivo have not yet been extensively studied, DNA synthesis associated with these three repair pathways is
predicted to be accurate, consistent with their known roles in suppressing damage-induced mutagenesis. Repairs
requiring filling gaps of one or a few nucleotides, such as “short patch” base excision repair and repair of DNA
double strand breaks by non-homologous end joining, use family X polymerases. On average, these polymerases
are less accurate than the main replicative polymerases (Table 2), partly but not exclusively due to lack of intrinsic

Table 2. Average single base substitution and deletion

error rates

Polymerase Proofreading Family Error rate (× 10−5 )

Subs. −1

Pol ε Yes B ≤1 ≤ 0.5

Pol δ Yes B ∼1 2
Pol γ Yes A ≤1 0.6
Pol α No B 16 3
Pol β No X 67 13
Pol λ No X 90 450
Pol κ No Y 580 180
Pol η No Y 3500 240
Pol ι No Y 72,000

The values for Pol ε and Pol δ are for calf thymus polymerases.
The values for the other polymerases are for human enzymes.
The Pol ι value is for T-dGTP mismatches only.
Pol ι error rates for other mismatches vary widely.

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Educational Session: Novel Drug Targets in DNA Replication

proofreading. It is possible that errors made by X family polymerases during repair or errors made by Y family
polymerases during TLS (see below) may be proofread by exonucleases intrinsic to other proteins, e.g., apurinic
endonuclease (10) or Pol ε or Pol δ (11). Nonetheless, the lower intrinsic fidelity of certain polymerases is likely
to reflect their normal in vivo functions. For example, the low single-base deletion fidelity of Pol λ indicates
the ability to use template-primers with minimal base pairing homology, consistent with its likely role in non-
homologous end joining of double strand breaks in DNA (12).
Lesions that escape repair can potentially reduce replication fidelity. TLS polymerases copy past lesions in
DNA that block the major replicative polymerases. One is the B family member Pol ζ , and others are in the Y
family (e.g., human Pol η, Pol ι, and Pol κ). Also lacking proofreading activity, Y family members are the least
accurate polymerases, with base substitution error rates when copying undamaged templates that generally range
from 10−1 to 10−3 (Table 2). The least accurate of these is Pol ι, which inserts dGTP opposite template T even
more efficiently than it inserts A opposite T, i.e., its error rate for this mispair approaches one. Individual error
rates for the 12 possible single base-base mismatches and for single-base misalignments differ widely depending
not only on the DNA polymerase but also on the composition of the mismatch and the sequence surrounding
the mismatch. These variables potentially allow extraordinary variation (e.g., see Fig.1 in Ref. 2) in the rate at
which DNA synthesis in human cells generates single base substitution and deletion error rates. High fidelity is
important for genome stability, but low fidelity can also be beneficial, e.g., for phase variation at contingency loci
in certain microbes or for generating base substitution errors during somatic hypermutation of immunoglobulin
genes (4).
Because the fidelity of accurate DNA polymerases depends on selection for correct Watson-Crick base pairing
geometry in the polymerase active site (2), the much lower fidelity of Y family polymerases most likely reflects
their biological roles in bypassing lesions that distort helix geometry, such as UV photoproducts and adducts
form by chemical modifications. The importance of TLS is clearly revealed by the increased susceptibility to
sunlight-induced skin cancer seen in Xeroderma pigmentosum patients harboring mutations in the POLH (XPV)
gene that inactivate Pol η. This phenotype is thought to reflect the ability of Pol η to efficiently copy DNA
templates containing a cis-syn cyclobutane dimer (CPD), one of several major lesions generated by exposure to
sunlight. The current hypothesis (13) is that, unlike certain other UV photoproducts, CPDs are slowly repaired
and therefore encountered by the replication machinery. CPDs block DNA synthesis by accurate replicative
polymerases, while Pol η efficiently bypasses them while avoiding generating CPD-induced mutations. In
the absence of Pol η, CPDs are bypassed in a less accurate manner by a different polymerase(s), generating
mutations that lead to skin cancer. This strong association between loss of Pol η activity and skin cancer has
led to bypass studies of a wide variety of other lesions by numerous polymerases. Among these, the product of
the REV3 gene (Pol ζ ) is clearly an important determinant of mutagenesis induced by several DNA damaging
agents (3).

Future Directions
Several issues deserve further investigation in order to address possible relationships between DNA polymerase
dysfunctions and cancer. Additional TLS studies are justified, especially using approaches that quantify relative
lesion bypass efficiency and the fidelity of complete lesion bypass among the large number of enzyme that
potentially might compete for bypass. Especially relevant will be studies to determine how polymerase switching
is coordinated both before and after lesion bypass, e.g., as determined by the intrinsic differences in substrate
preference by different polymerases, by replication accessory proteins, and by post-translational modification of
such proteins, e.g., PCNA (3). A related issue is how low fidelity polymerases are prevented from potentially
mutagenic and carcinogenic synthesis when not needed. Control could be exerted post-translationally or tran-
scriptionally, perhaps in a tissue-specific manner. These issues and cancer susceptibility is being investigated in
mouse models, as exemplified by the Pol δ proofreading deficient mouse mentioned above (9). To date, knocking

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The Functions and Fidelity of Human DNA Polymerases

out Pol β and Pol ζ has lead to embryonic lethality, but additional studies with mice harboring strategic missense
mutations in these essential genes are needed. Mice knocked out for Pols κ, ι, λ, and µ are viable and fertile,
but as yet they have not been reported to exhibit predisposition to cancer (3, 7). Further studies are needed to
look for phenotypes that may appear after several generations and to look for cancer in response to environ-
mental stress. The fact that there are multiple members of family A, B, X and Y, and that they share common
properties, suggests functional redundancy in repair and TLS pathways. Thus, defective polymerase-dependent
cancer susceptibility may eventually be revealed in mice defective in multiple polymerases, or with a polymerase
defect and defects in other genes that influence genome stability, e.g., MMR or other damage response genes.
It will also be interesting to search for putative functional polymorphisms in human DNA polymerase genes
that might contribute to cancer susceptibility, perhaps synergistically with defects in other genes whose products
function in the many DNA transactions that determine genome stability. Finally, the fact that several of the
polymerases listed in Table 1 participate in cellular responses that increase cell survival following DNA damage
by chemotherapeutic agents justifies continued efforts to identify potent and highly specific DNA polymerase
inhibitors. This objective may be facilitated by high-resolution structural information on DNA polymerases
bound to their cognate substrates.

References 9. Goldsby RE, Hays LE, Chen X, et al. High incidence of epithelial cancers
1. Friedberg EC, Walker GC, Siede W. In: DNA Repair and Mutagenesis. in mice deficient for DNA polymerase δ proofreading. Proc Natl Acad Sci
Washington, DC: American Society of Microbiology, 1995. USA 2002;99:15560–5.

2. Kunkel TA. DNA replication fidelity. J Biol Chem 2004;279:16895–98.
3. Plotsky BS, Woodgate R. Switching from high-fidelity replicases to low
fidelity lesion-bypass polymerases. Curr Opin Genet Devel 2004;14:113–9.
4. Kunkel TA, Erie D. DNA Mismatch Repair. Annu Rev Biochem 2005;in
press.
5. Muller A, Fishel R. Mismatch repair and the hereditary non-polyposis
colorectal cancer syndrome (HNPCC). Cancer Invest 2002;20:102–9.
6. Jiricny J, Marra G. DNA repair defects in colon cancer. Curr Opin Genet
Dev 2003;13:61–9.
7. Bebenek K, Kunkel TA. Functions of DNA polymerases. Adv Prot Chem
2004;69;137–65.
8. Kunkel TA. Considering the cancer consequences of altered DNA poly-
merase function. Cancer Cell 2003;3,105–10.
10. Matsuda T, Vande Berg BJ, Bebenek K, Osheroff WP, Wilson SH,
Kunkel TA. The base substitution fidelity of DNA polymerase β-dependent
single-nucleotide base excision repair. J Biol Chem. 2003;278:25947–
51.
11. McCulloch SD, Kokoska RJ, Chilkova O. et al. Enzymatic switching
for efficient and accurate translesion DNA replication. Nucl Acids Res
2004;32:4665–75.
12. Garcia-Diaz M, Bebenek K, Krahn JM, Blanco L, Kunkel TA, Ped-
ersen LC. A structural solution for the DNA polymerase λ-dependent
repair of DNA gaps with minimal homology. Molec Cell 2004:13:561–
72.
13. Kunkel TA, Pavlov YI, Benenek K. Functions of human DNA poly-
merases η, κ and ι suggested by their properties, including fidelity with
undamaged DNA templates. DNA Repair 2003;2:135–49.

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