Error-prone DNA Polymerase Research Articles

Dual role of NER in mutagenesis in Pseudomonas putida

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida. The genome of this organism contains two uvrA genes, uvrA and uvrA2. Genetic studies on the effects of uvrA, uvrA2, uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida. However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB, uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2, uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida—besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.

The microbiology of mutability

Bacteria possessing elevated spontaneous mutation rates are prevalent in certain environments, which is a paradox because most mutations are deleterious. For example, cells with defects in the methyl-directed mismatch repair (MMR) system, termed mutators or hypermutators, are overrepresented in populations of bacterial pathogens, with the mutator trait hypothesized to be advantageous in the changing host enviroments faced during colonization and establishment of chronic infections. Error-prone DNA polymerases, such as polIV and polV, function in translesion DNA synthesis, a DNA damage response that ensures genome integrity with a cost of increased mutation. While the biochemical aspects of these mutability pathways are well understood, the biological impacts have received less attention. Here, an examination of bacterial mutability systems and specifically the ecological and evolutionary context resulting in the selection of these systems is carried out.

Oxidative DNA Damage Defense Systems in Avoidance of Stationary-Phase Mutagenesis in Pseudomonas putida

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria.

2-Hydroxy-2‘-deoxyadenosine 5‘-Triphosphate Enhances A·T → C·G Mutations Caused by 8-Hydroxy-2‘-deoxyguanosine 5‘-Triphosphate by Suppressing Its Degradation upon Replication in a HeLa Extract

The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.

Use of Damaged DNA and dNTP Substrates by the Error-Prone DNA Polymerase X from African Swine Fever Virus

The structural specificity that translesion DNA polymerases often show for a particular class of lesions suggests that the predominant criterion of selection during their evolution has been the capacity for lesion tolerance and that the error-proneness they display when copying undamaged templates may simply be a byproduct of this adaptation. Regardless of selection criteria/evolutionary history, at present both of these properties coexist in these enzymes, and both properties confer a fitness advantage. The repair polymerase, Pol X, encoded by the African swine fever virus (ASFV) is one of the most error-prone polymerases known, leading us to previously hypothesize that it may work in tandem with the exceptionally error-tolerant ASFV DNA ligase to effect viral mutagenesis. Here, for the first time, we test whether the error-proneness of Pol X is coupled with a capacity for lesion tolerance by examining its ability to utilize the types of damaged DNA and dNTP substrates that are expected to be relevant to ASFV. We (i) test Pol X's ability to both incorporate opposite to and extend from ubiquitous oxidative purine (7,8-dihydro-8-oxoguanine), oxidative pyrimidine (5,6-dihydroxy-5,6-dihydrothymine), and noncoding (AP site) lesions, in addition to 5,6-dihydrothymine, (ii) determine the catalytic efficiency and dNTP specificity of Pol X when catalyzing incorporation opposite to, and when extending from, 7,8-dihydro-8-oxoguanine in a template/primer context, and (iii) quantitate Pol X-catalyzed incorporation of the damaged nucleotide 8-oxo-dGTP opposite to undamaged templates in the context of both template/primer and a single-nucleotide gap. Our findings are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.

Upregulation of Error-Prone DNA Polymerases Beta and Kappa Slows Down Fork Progression Without Activating the Replication Checkpoint

There is rising evidence that cancer development is associated from its earliest stages with DNA replication stress, a major source of spontaneous genomic instability. However, the origin of these replication defects has remained unclear. We have investigated the consequences of upregulating error-prone DNA polymerases (pol) beta and kappa on chromosomal DNA replication. These enzymes are misregulated in different types of cancers and induce major chromosomal instabilities when overexpressed at low levels. Here, we have used DNA combing to show that a moderate overexpression of pol beta or pol kappa is sufficient to impede replication fork progression and to promote the activation of additional replication origins. Interestingly, alterations of the normal replication program induced by excess error-prone polymerases were not detected by the replication checkpoint. We therefore propose that upregulation of error-prone DNA polymerases induces a checkpoint-blind replication stress that contributes to genomic instability and to cancer development.

Stress-Induced Mutagenesis in Bacteria

ABSTRACTBacteria spend their lives buffeted by changing environmental conditions. To adapt to and survive these stresses, bacteria have global response systems that result in sweeping changes in gene expression and cellular metabolism. These responses are controlled by master regulators, which include: alternative sigma factors, such as RpoS and RpoH; small molecule effectors, such as ppGpp; gene repressors such as LexA; and, inorganic molecules, such as polyphosphate. The response pathways extensively overlap and are induced to various extents by the same environmental stresses. These stresses include nutritional deprivation, DNA damage, temperature shift, and exposure to antibiotics. All of these global stress responses include functions that can increase genetic variability. In particular, up-regulation and activation of error-prone DNA polymerases, down-regulation of error-correcting enzymes, and movement of mobile genetic elements are common features of several stress responses. The result is that under a variety of stressful conditions, bacteria are induced for genetic change. This transient mutator state may be important for adaptive evolution.

A Role for PCNA Ubiquitination in Immunoglobulin Hypermutation

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNAK164R mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.

PBP3 inhibition elicits adaptive responses in Pseudomonas aeruginosa

Adaptive evolution depends on both the genetic variability in a population of organisms and the selection of the better adapted genotypes. However, for the fittest variants to be selected they must survive over a sufficient period under the new conditions. Bacteria are often exposed to different types of stress in nature, including antibiotics. We analysed the global expression profiles of the opportunistic pathogen Pseudomonas aeruginosa in response to ceftazidime, a PBP3 inhibitor, at different concentrations and times. PBP3 inhibition exerts a global impact on the transcription of a large number of genes. From an adaptive perspective, it is noteworthy the induction of several SOS genes, as well as adaptation, protection and antibiotic resistance genes. Intriguingly, transcription of pyocin genes, previously described as SOS-regulated, was repressed upon PBP3 inhibition. Ciprofloxacin, an SOS inducer, produced transcriptional induction of pyocins. Our results indicate that: (i) the SOS responses resulting from treatments with these two antibiotics cause only partially overlapping transcription profiles; (ii) PBP3 and DNA-gyrase inhibition produce opposite effects on transcription of pyocin genes. Consequently, ceftazidime decreases ciprofloxacin toxicity; (iii) error-prone DNA-polymerase DinB is induced by PBP3 inhibition but not by DNA-gyrase inhibition; (iv) PBP3 inhibition causes induced mutagenesis; (v) ceftazidime upregulates several antibiotic-resistance and adaptation genes; and (vi) ceftazidime concentrations thought previously to be lethal are not, as most cells treated with ceftazidime remain alive and recover their capacity to form colonies. Thus, transcriptional changes demonstrated in this work are likely to be adaptively relevant to cells that survive.

The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G 2 /M phase rather than S phase

The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase zeta (Rev3/7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase zeta are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein-protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the C-terminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are approximately 50-fold higher in G(2) and throughout mitosis than during G(1) and much of S phase. Differential survival of a rev1Delta strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.

First AID (Activation-induced Cytidine Deaminase) Is Needed to Produce High Affinity Isotype-switched Antibodies

An answer to the question "why don't you die when I sneeze?" resides in the ability of higher organisms to mount an aggressive defense in response to an invasion by foreign substances called antigens. In part this defense mechanism is accomplished through the synthesis of high affinity antibody (Ab) 2 proteins that effectively defuse the attack. Initially, lymphoid B cells rearrange immunoglobulin (Ig) genes using V(D)J recombination to generate a diverse repertoire of low affinity Abs. Subsequently these Ig genes undergo somatic hypermutation (SHM) and class switch recombination (CSR) to generate high affinity Ab molecules of different isotypes. Activation-induced cytidine deaminase (AID) is required to initiate both CSR and SHM. The current understanding of immunological diversification can be attributed to the contributions made by cellular and molecular immunologists with biochemical enzymology recently entering the arena. This minireview discusses advances in the understanding ofAb diversification from a biochemical perspective emphasizing the enzymatic properties of AID in initiating SHM and CSR and exploring the role of downstream mutation fixation events mediated by mismatch repair (MMR), basic excision repair (BER), and error-prone DNA polymerases (EP pols).

Effect of subinhibitory concentrations of antibiotics on mutation frequency in Streptococcus pneumoniae

To investigate the effect of subinhibitory concentrations of ciprofloxacin, streptomycin, trimethoprim, ampicillin and erythromycin on mutation frequency in Streptococcus pneumoniae. Frequency of mutation to rifampicin resistance was determined in three clinical isolates grown with or without antibiotic treatment. dinB was analysed using PCR and DNA sequence determination. Subinhibitory levels of ciprofloxacin and streptomycin increased the frequency of mutation to rifampicin resistance between 2- and 5-fold for all three isolates, which is comparable to the increase seen in mismatch repair mutants of this species. These increases appeared not to be dependent on the function of the error-prone DNA polymerase encoded by dinB, since one of the isolates was a naturally occurring deletion mutant for this gene. Trimethoprim increased the mutation frequency for two isolates, but not the dinB mutant; ampicillin and erythromycin had no significant effect on mutation frequencies for any isolate. Exposure to quinolones and aminoglycosides at subinhibitory concentrations may result in increased mutability in pneumococci, as well as selecting for resistance per se.

Translesion DNA synthesis across non-DNA segments in cultured human cells

DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS), carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both the trimethylene and dodecamethylene inserts were bypassed with significant efficiencies in human cells, using both misinsertion and misalignment mechanisms. TLS across these non-DNA segments was aphidicolin-sensitive, and did not require polη. In vitro primer extension assays showed that purified polη, polκ and polι were each capable of inserting each of the four nucleotides opposite the trimethylene chain, but only polη and polκ could fully bypass it. Polη and polι, but not polκ, could also insert each of the four nucleotides opposite the dodecamethylene chain, but all three polymerases were severely blocked by this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of transient and local template-independent polymerase activity, and highlights the robustness of the TLS system in human cells.

The role of DNA polymerase ι in UV mutational spectra

UVB (280–320 nm) and UVC (200–280 nm) irradiation generate predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine–thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase η (Pol η) dependent process. Pol η is a DNA damage-tolerant and error-prone DNA polymerase encoded by the POLH ( XPV) gene in humans. Another member of the Y family of error-prone DNA polymerases is POLI encoding DNA polymerase iota (Pol ι). In order to clarify the specific role of Pol ι in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells, similar as we have previously done for Pol η. Synthetic RNA duplexes were used to efficiently inhibit Pol ι expression in 293T cells. The supF shuttle vector was irradiated with 254 nm UVC and replicated in 293T cells in presence of anti-Pol ι siRNA. Surprisingly, there was a consistent reduction of recovered plasmid from cells with Pol ι knockdown and this was independent of UV irradiation of the plasmid. The supF mutant frequency was unchanged in the siRNA knockdown cells relative to control cells confirming that Pol ι does not play an important role in UV mutagenesis. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Neither the type of mutations nor their distribution along the supF gene were significantly different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. These results show that Pol ι has no significant role in UV lesion bypass and mutagenesis in vivo and provides some initial data suggesting that this polymerase may be involved in replication of extrachromosomal DNA.

Mechanistic Comparison of High-Fidelity and Error-Prone DNA Polymerases and Ligases Involved in DNA Repair

Mechanistic Comparison of High-Fidelity and Error-Prone DNA Polymerases and Ligases Involved in DNA Repair

Transcriptional Control of Activation-Induced Cytidine Deaminase and Error-Prone DNA Polymerases Is Functionally Mature in the B Cells of Infants at Birth

Somatic hypermutation (SHM) of immunoglobulin genes requires activation-induced cytidine deaminase (AID). The error-prone DNA polymerases, such as Pol η, Pol ζ, and Pol ι, also have been implicated in the process. Human adult antibodies directed to microbial pathogens are increased in affinity and function compared with those of infants. Adult antibodies achieve this increased affinity through somatic mutations, which are lacking in the B cells of infants. It is unknown if infant B cells are capable of upregulating the cell machinery needed to introduce mutations after stimulation through the antigen receptor. We show here that infant B cells exhibit similar kinetics and magnitude of transcription of AID and pol η genes and only marginally lower levels of pol ι and pol ζ genes after stimulation through the B cell receptor. These data suggest that the ability to upregulate gene transcription of enzymes mediating SHM is not a limiting determinant of the functional quality of infant antibody responses.

Eukaryotic error-prone DNA polymerases: The presumed roles in replication, repair, and mutagenesis

A number of error-prone DNA polymerases is found among eukaryotes from yeasts up to mammalia including humans. According to the partial homology of a primary structure, they are united in families B, X, Y and display high infidelity on uninjured DNA-template, whereas they are rather accurate on DNA injuries. These DNA polymerases are characterized by the probability of base substitutions or frame shifts of 10(-3) to 7.5 x 10(-1) on DNA injuries, whereas the probability of spontaneous mutagenesis per replicated nucleotide accounts 10(-10) - 10(-12). Inaccurate DNA polymerases are terminal deoxynucleotidyl transferase (TdT), DNA polymerases beta, zeta, kappa, eta, iota, lamda, mu, and Rev1. Their principal properties are described in this review. All of the polymerases under study are deprived of the corrective 3'-->5' exonucleolytic activity. The specialization of these polymerases is contained in the capability to synthesize opposite DNA lesions (not eliminated by multiple repair systems) that is explained by the flexibility of their active sites or by the limited capability to exhibit the TdT activity. Classic DNA polymerases alpha, delta, epsilon, and gamma cannot elongate the primers with mismatched nucleotides on their 3'-ends (that leads to the replication block), whereas some of the specialized polymerases can do it. It is accompanied by the overcoming of a replication block, often with the expense of an elevated mutagenesis. How can a cell live under the conditions of such a huge infidelity of many DNA polymerases? Error-prone DNA polymerases are not found in all tissues though some of them are essential for an organism survival. Furthermore, cells must not allow for these polymerases to work effectively on uninjured DNA. After bypass of a lesion on DNA-template, it is necessary, as soon as possible, to switch catalysis of the DNA synthesis from the specialized polymerases on the relatively accurate DNA polymerases delta and epsilon (fidelity of 10(-5) - 10(-6)). It is made by the formation of the complexes of polymerases delta or epsilon with PCNA and replicative factors RP-A and RF-C. Such highly processive complexes manifest the bigger affinity to the correct primers than the specialized DNA polymerases do. The switching is stimulated by distributivity or weak processivity of the specialized DNA polymerases. The accuracy of these polymerases are augmented by the action of the corrective 3'-exonucleolytic function of DNA polymerases delta and epsilon as well as by the autonomous 3'-->5' exonucleases which are widespread among the representatives of the whole phylogenetic tree. Exonucleolytic correction slows down the replication in the presence of lesions in DNA-template but makes the replication more accurate that decreases the probability of mutagenesis and carcinogenesis.

UV Sensitivity and Mutagenesis of the Extremely Thermophilic Eubacterium Thermus thermophilus HB27

Thermus thermophilus is an aerobic, extremely thermophilic eubacterium that grows optimally at 70-75°C. Phylogenetically there is a close relationship between T. thermophilus and Deinococcus radiodurans, an extraordinary radiation-resistant bacterium. In contrast to the D. radiodurans, DNA repair and mutagenesis in T. thermophilus have not been investigated intensively. DNA repair and related mutagenesis at high temperature are particularly interesting because the frequency of DNA damage, such as deamination, depurination, and single strand breaks, is expected to be greatly increased. We disrupted the uvrA gene for subunit A of excinuclease ABC and uvdE gene for probable UV endonuclease by inserting the thermostable kanamycin-resistant gene (HTK) or orotate phosphoribosyltransferase (pyrE) gene. A uvrA mutant showed moderate sensitivity to UV-irradiation, while a uvdE mutant was not UV sensitive. A uvdE uvrA double mutant was highly sensitive to UV-irradiation compared with a uvrA mutant, indicating that UV endonuclease, a uvdE gene product, plays an important role in repair of UV-induced DNA damages. On the other hand, no obvious UV-induced mutations were observed when it was assayed by His+ reversion. It has been reported that T. thermophilus HB27 does not have an SOS response system, because no genes homologous to umuD, umuC, and lexA were found. In the absence of error-prone translesion DNA polymerases in T. thermophilus, mutations would not occur at the site of pyrimidine dimers

Roles of E. coli double-strand-break-repair proteins in stress-induced mutation

Special mechanisms of mutation are induced during growth-limiting stress and can generate adaptive mutations that permit growth. These mechanisms may provide improved models for mutagenesis in antibiotic resistance, evolution of pathogens, cancer progression and chemotherapy resistance. Stress-induced reversion of an Escherichia coli episomal lac frameshift allele specifically requires DNA double-strand-break-repair (DSBR) proteins, the SOS DNA-damage response and its error-prone DNA polymerase, DinB. We distinguished two possible roles for the DSBR proteins. Each might act solely upstream of SOS, to create single-strand DNA that induces SOS. This could upregulate DinB and enhance mutation globally. Or any or all of them might function other than or in addition to SOS promotion, for example, directly in error-prone DSBR. We report that in cells with SOS genes derepressed constitutively, RecA, RuvA, RuvB, RuvC, RecF, and TraI remain required for stress-induced mutation, demonstrating that these proteins act other than via SOS induction. RecA and TraI also act by promoting SOS. These and additional results with hyper-mutating recD and recG mutants support roles for these proteins via error-prone DSBR. Such mechanisms could localize stress-induced mutagenesis to small genomic regions, a potentially important strategy for adaptive evolution, both for reducing additional deleterious mutations in rare adaptive mutants and for concerted evolution of genes.

Translesion DNA replication proteins as molecular targets for cancer prevention

Mutations in DNA are generally considered to have an etiologic role in the development of cancer. If so, it follows that reducing the frequency of such mutations will reduce the incidence of cancer induced by mutagens. Recent advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements, and can insert bases across from adducted templates, but with potentially mutagenic consequences. In principle, these proteins present new and attractive molecular targets to reduce mutagenesis. If this can be done in vivo without increasing cytotoxic responses to carcinogens, then novel chemopreventive strategies can be designed to reduce the risk of cancer in exposed populations prior to the appearance of disease symptoms.