Genetic requirement for mutagenesis of the G[8,5-Me]T cross-link in Escherichia coli: DNA polymerases IV and V compete for error-prone bypass

γ-Radiation generates a variety of complex lesions in DNA, including the G[8,5-Me]T intrastrand cross-link in which C8 of guanine is covalently linked to the 5-methyl group of the 3'-thymine. We have investigated the toxicity and mutagenesis of this lesion by replicating a G[8,5-Me]T-modified plasmid in Escherichia coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these DNA polymerases. In the single-polymerase knockout strains, viability was the lowest in a pol V-deficient strain, which suggests that pol V is most efficient in bypassing this lesion. Most mutations were single-base substitutions or deletions, though a small population of mutants carrying two point mutations at or near the G[8,5-Me]T cross-link was also detected. Mutations in the progeny occurred at the cross-linked bases as well as at bases near the lesion site, but the mutational spectrum varied on the basis of the identity of the DNA polymerase that was knocked out. Mutation frequency was the lowest in a strain that lacked the three SOS DNA polymerases. We determined that pol V is required for most targeted G → T transversions, whereas pol IV is required for the targeted T deletions. Our results suggest that pol V and pol IV compete to carry out error-prone bypass of the G[8,5-Me]T cross-link.     

Human mitochondrial DNA polymerase γ exhibits potential for bypass and mutagenesis at UV-induced cyclobutane thymine dimers

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3'-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3'-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.     

Effects of the C4'-oxidized abasic site on replication in Escherichia coli. An unusually large deletion is induced by a small lesion

The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress by a variety of agents. For instance, the lesion accounts for approximately 40% of the DNA damage produced by the antitumor antibiotic bleomycin. The effect of C4-AP on DNA replication in Escherichia coli was determined using the restriction endonuclease and postlabeling (REAP) method. Three-nucleotide deletion products are the sole products observed following replication of plasmids containing C4-AP under SOS conditions in wild-type cells. Full-length products are formed in varying amounts depending upon the local sequence in wild-type cells under non-SOS-induced conditions. The "A-rule" is followed for the formation of substitution products. C4-AP is the first example of a DNA lesion that produces significant levels of three-nucleotide deletions in a variety of sequence contexts. Experiments carried out in cells lacking specific polymerases reveal that formation of three-nucleotide deletion products results from a coordinated effort involving pol II and pol IV. This is the first example in which these SOS inducible polymerases are shown to work in concert during lesion bypass. Three-nucleotide deletions are not observed during the replication of other abasic lesions, and are rarely produced by bulky adducts. The effect of C4-AP on DNA replication suggests a significant role for this lesion in the cytotoxicity of bleomycin. Formation of the C4-AP lesion may also be responsible for the formation of mutant proteins containing single-amino acid deletions that exhibit altered phenotypes.     

All three SOS-inducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis

Most organisms contain several members of a recently discovered class of DNA polymerases (umuC/dinB superfamily) potentially involved in replication of damaged DNA. In Escherichia coli, only Pol V (umuDC) was known to be essential for base substitution mutagenesis induced by UV light or abasic sites. Here we show that, depending upon the nature of the DNA damage and its sequence context, the two additional SOS-inducible DNA polymerases, Pol II (polB) and Pol IV (dinB), are also involved in error-free and mutagenic translesion synthesis (TLS). For example, bypass of N:-2-acetylaminofluorene (AAF) guanine adducts located within the NAR:I mutation hot spot requires Pol II for -2 frameshifts but Pol V for error-free TLS. On the other hand, error-free and -1 frameshift TLS at a benzo(a)pyrene adduct requires both Pol IV and Pol V. Therefore, in response to the vast diversity of existing DNA damage, the cell uses a pool of 'translesional' DNA polymerases in order to bypass the various DNA lesions.     

Analysis of translesion replication across an abasic site by DNA polymerase IV of Escherichia coli

Unrepaired replication-blocking DNA lesions are bypassed by specialized DNA polymerases, members of the Y super-family. In Escherichia coli the major lesion bypass DNA polymerase is pol V, whereas the function of its homologue, pol IV, is not fully understood. In vivo analysis showed that pol V has a major role in bypass across an abasic site analog, with little or no involvement of pol IV. This can result from the inability of pol IV to bypass the abasic site, or from in vivo regulation of its activity. In vitro analysis revealed that purified pol IV, in the presence of the beta subunit DNA sliding clamp, and the gamma complex clamp loader, bypassed a synthetic abasic site with very high efficiency, reaching 73% in 2 min. Bypass was observed also in the absence of the processivity proteins, albeit at a 10- to 20-fold lower rate. DNA sequence analysis revealed that pol IV skips over the abasic site, producing primarily small deletions. The RecA protein inhibited bypass by pol IV, but this inhibition was alleviated by single-strand binding protein (SSB). The fact that the in vitro bypass ability of pol IV is not manifested under in vivo conditions suggests the presence of a regulatory factor, which might be involved in controlling the access of the bypass polymerases to the damaged site in DNA.     

Roles of replicative and specialized DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains lacking one or all of SOS-inducible DNA polymerases to 26 chemicals

Progression of DNA replication is occasionally blocked by endogenous and exogenous DNA damage. To circumvent the stalling of DNA replication, cells possess a variety of specialized DNA polymerases that replicate through DNA damage. Salmonella typhimurium strain TA1538 has six DNA polymerases and four of them are encoded by damage-inducible SOS genes, i.e. polB(ST) (pol II), dinB(ST) (pol IV), umuDC(ST) (pol V) and samAB. The strain has been used for the detection of a variety of chemical mutagens because of the high sensitivity to -2 frameshift occurring in CGCGCGCG sequence. To assign the role of each DNA polymerase in the frameshift mutagenesis, we have constructed the derivatives lacking one or all of SOS-inducible DNA polymerases and examined the mutability to 26 chemical mutagens. Interestingly, the chemicals could be categorized into four classes: class I whose mutagenicity was reduced by the deletion of dinB(ST) (1-aminoanthracene and other four chemicals); class II whose mutagenicity was reduced by the deletion of either dinB(ST) or umuDC(ST) plus samAB (7,12-dimethylbenz[a]anthracene and other three chemicals); class III whose mutagenicity largely depended on the presence of umuDC(ST) plus samAB (1-N-6-azabenzo[a]pyrene and other three chemicals) and class IV whose mutagenicity was not reduced by deletion of any of the genes encoding SOS-inducible DNA polymerases (Glu-P-1 and other 12 chemicals). Deletion of polB(ST) reduced by 30-60% the mutagenicity of six chemicals of classes II and III. These results suggest that multiple DNA polymerases including the replicative DNA polymerase, i.e. DNA polymerase III holoenzyme, play important roles in chemically induced -2 frameshift and also that different sets of DNA polymerases are engaged in the translesion bypass of different DNA lesions.     

Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways

Living cells possess a panel of specialized DNA polymerases that deal with the large diversity of DNA lesions that occur in their genomes. How specialized DNA polymerases gain access to the replication intermediate in the vicinity of the lesion is unknown. Using a model system in which a single replication blocking lesion can be bypassed concurrently by two pathways that leave distinct molecular signatures, we analyzed the complex interplay among replicative and specialized DNA polymerases. The system involves a single N-2-acetylaminofluorene guanine adduct within the NarI frameshift hot spot that can be bypassed concurrently by Pol II or Pol V, yielding a −2 frameshift or an error-free bypass product, respectively. Reconstitution of the two pathways using purified DNA polymerases Pol III, Pol II and Pol V and a set of essential accessory factors was achieved under conditions that recapitulate the known in vivo requirements. With this approach, we have identified the key replication intermediates that are used preferentially by Pol II and Pol V, respectively. Using single-hit conditions, we show that the β-clamp is critical by increasing the processivity of Pol II during elongation of the slipped −2 frameshift intermediate by one nucleotide which, surprisingly, is enough to support subsequent elongation by Pol III rather than degradation. Finally, the proofreading activity of the replicative polymerase prevents the formation of a Pol II-mediated −1 frameshift product. In conclusion, failure or success of TLS pathways appears to be the net result of a complex interplay among DNA polymerases and accessory factors.     

A dynamic polymerase exchange with Escherichia coli DNA polymerase IV replacing DNA polymerase III on the sliding clamp

An assay that measures synchronized, processive DNA replication by Escherichia coli DNA polymerase III holoenzyme was used to reveal replacement of pol III by the specialized lesion bypass DNA polymerase IV when the replicative polymerase is stalled. When idled replication is restarted, a rapid burst of pol III-catalyzed synthesis accompanied by approximately 7-kb full-length products is strongly inhibited by the presence of pol IV. The production of slower-forming, shorter length DNA reflects a rapid takeover of DNA synthesis by pol IV. Here we demonstrate that pol IV rapidly (<15 s) obstructs the stable interaction between pol III* and the beta clamp (the lifetime of the complex is >5 min), causing the removal of pol III* from template DNA. We propose that the rapid replacement of pol III* on the beta clamp with pol IV is mediated by two processes, an interaction between pol IV and the beta clamp and a separate interaction between pol IV and pol III*. This newly discovered property of pol IV facilitates a dynamic exchange between the two free polymerases at the primer terminus. Our study suggests a model in which the interaction between pol III* and the beta clamp is mediated by pol IV to ensure that DNA replication proceeds with minimal interruption.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.     

Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli

The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.     

Multiple two-polymerase mechanisms in mammalian translesion DNA synthesis

The encounter of replication forks with DNA lesions may lead to fork arrest and/or the formation of single-stranded gaps. A major strategy to cope with these replication irregularities is translesion DNA replication (TLS), in which specialized error-prone DNA polymerases bypass the blocking lesions. Recent studies suggest that TLS across a particular DNA lesion may involve as many as four different TLS polymerases, acting in two-polymerase reactions in which insertion by a particular polymerase is followed by extension by another polymerase. Insertion determines the accuracy and mutagenic specificity of the TLS reaction, and is carried out by one of several polymerases such as polη, polκ or polι. In contrast, extension is carried out primarily by polζ. In cells from XPV patients, which are deficient in TLS across cyclobutane pyrimidine dimers (CPD) due to a deficiency in polη, TLS is carried out by at least two backup reactions each involving two polymerases: One reaction involves polκ and polζ, and the other polι and polζ. These mechanisms may also assist polη in normal cells under an excessive amount of UV lesions.     

The N-clasp of human DNA polymerase kappa promotes blockage or error-free bypass of adenine- or guanine-benzo[a]pyrenyl lesions

DNA bypass polymerases are utilized to transit bulky DNA lesions during replication, but the process frequently causes mutations. The structural origins of mutagenic versus high fidelity replication in lesion bypass is therefore of fundamental interest. As model systems, we investigated the molecular basis of the experimentally observed essentially faithful bypass of the guanine 10S-(+)-trans-anti-benzo[a]pyrene-N²-dG adduct by the Y-family human DNA polymerase κ, and the observed blockage of pol κ produced by the adenine 10S-(+)-trans-anti-benzo[a]pyrene-N²-dA adduct. These lesions are derived from the most tumorigenic metabolite of the ubiquitous cancer-causing pollutant, benzo[a]pyrene. We compare our results for the dG adduct with our earlier studies for the pol κ archaeal homolog Dpo4, which processes the same lesion in an error-prone manner. Molecular modeling, molecular mechanics calculations and molecular dynamics simulations were utilized. Our results show that the pol κ N-clasp is a key structural feature that accounts for the dA adduct blockage and the near-error-free bypass of the dG lesion. Absence of the N-clasp in Dpo4 explains the error-prone processing of the same lesion by this enzyme. Thus, our studies elucidate structure-function relationships in the fidelity of lesion bypass.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited

In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity. In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis. Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD'). In addition, RecA protein and the replication processivity factor, the beta-clamp, were genetically found to be essential co-factors for mutagenesis. Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements. Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the beta-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the beta-clamp and the 3' tip of the RecA filament. Formation of an extended RecA/ATP filament and stable loading of the beta-clamp are best achieved on long single-stranded circular DNA templates. In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPgammaS, activates the RecA filament. Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V.     

The spacious active site of a Y-family DNA polymerase facilitates promiscuous nucleotide incorporation opposite a bulky carcinogen-DNA adduct: elucidating the structure-function relationship through experimental and computational approaches

Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N(2)-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N(2)-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol kappa. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N(2)-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N(2)-dG adduct, albeit to a lesser extent than unmodified guanine, and the V(max) values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N(2)-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion.     

In vitro effects of a C4'-oxidized abasic site on DNA polymerases

Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites. The C4'-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents. This lesion accounts for approximately 40% of the DNA damage produced by bleomycin. The effect of a C4'-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3' --> 5' exonuclease activity. Both enzymes preferentially incorporated dA > dG > dC, T opposite C4-AP. Neither enzyme is able to extend the primer past the lesion. Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites. Extension of the primer by alternative polymerases pol II, pol II exo(-), pol IV, and pol V was examined. Pol II exo(-) was most efficient. Qualitative translesion synthesis experiments showed that pol II exo(-) preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC. Thymidine incorporation opposite C4'-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template. These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the "A-rule". The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C --> T:A transversions in cells treated with bleomycin.     

Two‐polymerase mechanisms dictate error‐free and error‐prone translesion DNA synthesis in mammals

DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error‐free, and the third slow and error‐prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase ζ (polζ), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two‐polymerase combinations with polζ dictate error‐prone or error‐free TLS across the same lesion. These results highlight the central role of polζ in both error‐prone and error‐free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two‐polymerase combinations.     

Nucleotide excision repair or polymerase V-mediated lesion bypass can act to restore UV-arrested replication forks in Escherichia coli

Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.     

Interplay of DNA repair,homologous recombination,and DNA polymerases in resistance to the DNA damaging agent 4-nitroquinoline-1-oxide in Escherichia coli

Escherichia coli has three DNA damage-inducible DNA polymerases: DNA polymerase II (Pol II), DNA polymerase IV (Pol IV), and DNA polymerase V (Pol V). While the in vivo function of Pol V is well understood, the precise roles of Pol IV and Pol II in DNA replication and repair are not as clear. Study of these polymerases has largely focused on their participation in the recovery of failed replication forks, translesion DNA synthesis, and origin-independent DNA replication. However, their roles in other repair and recombination pathways in E. coli have not been extensively examined. This study investigated how E. coli's inducible DNA polymerases and various DNA repair and recombination pathways function together to convey resistance to 4-nitroquinoline-1-oxide (NQO), a DNA damaging agent that produces replication blocking DNA base adducts. The data suggest that full resistance to this compound depends upon an intricate interplay among the activities of the inducible DNA polymerases and recombination. The data also suggest new relationships between the different pathways that process recombination intermediates.