DNA polymerase V allows bypass of toxic guanine oxidation products in vivo

Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.     

Evidence for Escherichia coli polymerase II mutagenic bypass of intrastrand DNA crosslinks

The mutagenic potentials of DNAs containing site- and stereospecific intrastrand DNA crosslinks were evaluated in Escherichia coli cells that contained a full complement of DNA polymerases or were deficient in either polymerases II, IV, or V. Crosslinks were made between adjacent N(6)-N(6) adenines and consisted of R,R- and S,S-butadiene crosslinks and unfunctionalized 2-, 3-, and 4-carbon tethers. Although replication of single-stranded DNAs containing the unfunctionalized 3- and 4-carbon tethers were non-mutagenic in all strains tested, replication past all the other intrastrand crosslinks was mutagenic in all E. coli strains, except the one deficient in polymerase II in which no mutations were ever detected. However, when mutagenesis was analyzed in cells induced for SOS, mutations were not detected, suggesting a possible change in the overall fidelity of polymerase II under SOS conditions. These data suggest that DNA polymerase II is responsible for the in vivo mutagenic bypass of these lesions in wild-type E. coli.     

Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells.

The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form. A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells. After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced. Our results show clearly that both lesions are mutagenic, but at different levels. Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T. With the T(6-4)T a large variety of mutations were observed. A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.     

SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells.

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.     

A comprehensive comparison of DNA replication past 2-deoxyribose and its tetrahydrofuran analog in Escherichia coli

Apurinic/apyrimidinic (AP) sites are alkali labile lesions that, when encountered during DNA replication, can block polymerases or potentially result in mutagenic events. Owing to the instability of 2-deoxyribose lesions (AP), a chemically stable tetrahydrofuran analog (F) is often used as a model of abasic sites. A comparison of the two lesions in Saccharomyces cerevisiae revealed that the model lesion and 2-deoxyribose have distinct in vivo effects. Comprehensive comparative analyses of F and AP have not been carried out in Escherichia coli. We conducted a side-by-side investigation of F and AP in E.coli to compare their biological effects and interactions with SOS polymerases. Both lesions were examined in SOS-induced and uninduced cells. Our studies reveal that in uninduced E.coli the effects of individual polymerases in the replication of plasmids containing F or AP are distinct. However, when cells are SOS-induced, the biological effects of F and AP are similar.     

Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III

Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature. The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell. In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.     

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences

Forward mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) in the tetracycline resistance gene carried on plasmid pBR322 are shown to be dependent upon the induction of the host SOS functions in wild-type and umuC Escherichia coli cells. The mutation frequency in the umuC strain is equal to about 40% of the mutation frequency observed in the umu+ background. In the excision-repair-deficient uvrA mutant strain the mutagenic response is the same as in SOS-induced wild-type cells whether or not the uvrA bacteria are SOS-induced. Equal mutation frequencies are obtained in both the wild-type and the uvrA strains for equal modification levels although the survival of AAF-modified plasmid DNA is greatly reduced in the uvrA strain as compared to the wild-type strain. Sequence analysis of the mutations reveals that more than 90% of the N-Aco-AAF-induced mutations are frameshift mutations. Two types of mutational hotspots are observed occurring either at repetitive sequences or at non-repetitive sequences. Both types of mutants appear at similar locations and frequencies in both the wild-type and the uvrA strains. On the other hand, only the non-repetitive sequence mutants are obtained in the umuC background. These non-repetitive sequence mutants preferentially occur within the sequence 5' G-G-C-G-C-C 3' (the NarI restriction enzyme recognition sequence). The analysis of the -AAF binding spectrum to the same DNA fragment shows that there is no direct correlation between the modification spectrum and the mutation spectrum. We suggest that certain sequences are "mutation-prone" in the sense that only these sequences can be efficiently mutated as the result of an active processing mediated by specific proteins. When a sequence is said to be mutation-prone it probably corresponds to a particular structure that is induced within this sequence as a result of the binding to the DNA of the mutagen. This sequence-specific conformational change is the substrate for the protein(s) that fixes the mutation. The mutagenic processing pathway(s) is part of the cellular response to DNA-damaging agents (the so-called SOS response). Two pathways for frameshift mutagenesis are suggested by the data: an umuC-dependent pathway, which is involved in the mutagenic processing of lesions within repetitive sequences; an umuC-independent pathway responsible for the fixation of mutations within specific non-repetitive sequences.     

Peroxynitrite-induced reactions of synthetic oligo 2'-deoxynucleotides and DNA containing guanine: formation and stability of a 5-guanidino-4-nitroimidazole lesion

Peroxynitrite is a strong oxidizing agent that is formed in the reaction of nitric oxide and superoxide anion. It is capable of oxidizing and nitrating a variety of biological targets including DNA, and these modifications may be responsible for a number of pathological conditions and diseases. A recent study showed that peroxynitrite reacts with 2',3',5'-tri-O-acetylguanosine to yield a novel compound, tri-O-acetyl-1-(beta-D-erythro-pentafuranosyl)-5-guanidino-4-nitroimidazole, and, unlike other peroxynitrite-mediated guanine oxidation products, it is a stable and significant component formed even at low peroxynitrite concentrations. In this work, we studied the in vitro formation of the guanine-derived product, 5-guanidino-4-nitroimidazole, in synthetic oligonucleotides and DNA treated with peroxynitrite. When calf thymus DNA or oligonucleotides were reacted with peroxynitrite at ambient temperature, the modified base 5-guanidino-4-nitroimidazole was generated along with several other products. The oligonucleotides containing the 5-guanidino-4-nitroimidazole modification were purified by reverse-phase and anion-exchange HPLC and characterized by matrix-assisted laser desorption mass spectrometry. 5-Guanidino-4-nitroimidazole formation in peroxynitrite-treated DNA was characterized after enzymatic digestion of the reacted DNA to the nucleoside level. HPLC purification and electrospray ionization mass spectrometry (with selected reaction monitoring) enabled the analysis of this modified nucleoside with high sensitivity. The yield of 5-guanidino-4-nitroimidazole formed in single-stranded DNA was approximately 10-fold higher than that found in duplex DNA. With calf thymus DNA, 5-guanidino-4-nitroimidazole was dose-dependently formed at low peroxynitrite concentrations. In stability tests, a synthetic oligonucleotide containing the 5-guanidino-4-nitroimidazole modification was only partially cleaved by hot piperidine and was a weak substrate for Fpg glycosylase repair enzyme; in addition, this site was not cleaved by endonuclease III. These results suggest that nuclear DNA containing 5-guanidino-4-nitroimidazole may not be quickly repaired by DNA repair enzyme systems. Finally, primer extension experiments revealed that this lesion is a potential DNA replication blocker when polymerization is catalyzed by polymerase alpha and polymerase I (Klenow fragment, lack of exonuclease activity) but not with human polymerase beta. Replication fidelity experiments further showed that 5-guanidino-4-nitroimidazole may cause G-->T and G-->C transversions in calf thymus polymerase alpha and E. coli polymerase I.     

SOS-dependent A-->G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA.

Gas chromatography/isotope dilution-mass spectrometry with selected ion monitoring (GC/IDMS-SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C-->A (approximately 20-fold increase in SOS-conditions), G-->A (9-fold increase) and G-->C (4.5-fold increase). Very few G-->T transitions were found. A particularly large group of A-->G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A-->G transitions in SOS-induced bacteria.     

Specificity of mutagenesis resulting from the induction of the SOS system in the absence of mutagenic treatment

Strains in which the E. coli SOS system is continuously induced, in the absence of mutagenic treatment, have been used to generate nonsense mutations in the lacl gene. The examination of over 600 independently occurring amber and ochre mutations reveals that inducing the SOS system stimulates specifically G:C----T:A and, to a lesser extent, A:T----T:A transversions. This specificity is similar to that seen for a number of carcinogens that form bulky adducts to DNA, such as benzo(a)pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), and which are dependent on the SOS system to mutagenize bacteria. However, G:C----T:A transversions resulting from SOS induction alone display a unique site specificity. One possibility is that the SOS-induced mutations result from cryptic, spontaneous lesions, such as apurinic sites, which cannot induce the SOS system themselves but which can target mutations once the SOS system is induced.     

Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector.

We have investigated the mutagenic properties of an abasic site in DNA by transfecting SOS-induced and uninduced cells of E. coli with a single-stranded M13mp7-based vector that carries a single example of this lesion at one or other of two unique and adjacent sites. Random samples of progeny phage were sequenced to determine the nature of the replication events that occurred at and around these locations. 5% to 7% of the vectors could be replicated in SOS-induced cells, but only 0.1% to 0.7% of them gave plaques in the absence of SOS induction. In SOS-induced cells, 93% and 96% of the phage replicated resulted from the insertion of a nucleotide opposite the abasic site, while the remainder resulted from a targeted omission of a single nucleotide. At one of the sites, nucleotide insertions were 54% dAMP, 25% dTMP, 20% dGMP and 1% dCMP. At the other site they were 80% dAMP, 4% dTMP, 15% dGMP and 1% dCMP. The sequence variation in all but two of the 204 sequences analyzed was restricted to the abasic site itself. In the remaining two, a change at the abasic site was accompanied by a mutation at an immediately flanking nucleotide.     

Error-prone Replication Bypass of the Primary Aflatoxin B1 DNA Adduct,AFB1-N7-Gua

Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths worldwide. The highest rates of early onset HCCs occur in geographical regions with high aflatoxin B₁ (AFB₁) exposure, concomitant with hepatitis B infection. Although the carcinogenic basis of AFB₁ has been ascribed to its mutagenic effects, the mutagenic property of the primary AFB₁-DNA adduct, AFB₁-N7-Gua, in mammalian cells has not been studied extensively. Taking advantage of the ability to create vectors containing a site-specific DNA adduct, the mutagenic potential was determined in primate cells. This adduct was highly mutagenic following replication in COS-7 cells, with a mutation frequency of 45%. The spectrum of mutations was predominantly G to T base substitutions, a result that is consistent with previous mutation data derived from aflatoxin-associated HCCs. To assess which DNA polymerases (pol) might contribute to the mutational outcome, in vitro replication studies were performed. Unexpectedly, replicative pol δ and the error-prone translesion synthesis pol ζ were able to accurately bypass AFB₁-N7-Gua. In contrast, replication bypass using pol κ was shown to occur with low fidelity and could account for the commonly detected G to T transversions.     

The two-step model for translesion synthesis: then and now

The formation of base substitution mutations following exposure of bacteria to ultraviolet light and many other mutagens occurs during translesion synthesis opposite a photoproduct or other lesion in the template strand of DNA. This process requires the UmuD(2)' UmuC complex, only formed to a significant extent in SOS-induced cells. The "two-step" model proposed that there were two steps, insertion of a wrong base (misincorporation) and use of the misincorporated base as a primer for further chain extension (bypass). The original evidence suggested that UmuD(2)' UmuC was needed only for the second step and that in its absence other polymerases such as DNA polymerase III could make misincorporations. Now we know that the UmuD(2)' UmuC complex is DNA polymerase V and that it can carry out both steps in vitro and probably does both in vivo in wild-type cells. Even so, DNA polymerase III clearly has an important accessory role in vitro and a possibly essential role in vivo, the precise nature of which is not clear. DNA polymerases II and IV are also up-regulated in SOS-induced cells and their involvement in the broader picture of translesion synthesis is only now beginning to emerge. It is suggested that we need to think of the chromosomal replication factory as a structure through which the DNA passes and within which as many as five DNA polymerases may need to act. Protein-protein interactions may result in a cassette system in which the most appropriate polymerase can be engaged with the DNA at any given time. The original two-step model was very specific, and thus an oversimplification. As a general concept, however, it reflects reality and has been demonstrated in experiments with eukaryotic DNA polymerases in vitro.     

Error prone translesion synthesis past gamma-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells

8-Hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin- 10(3H)-one,3-(2'-deoxyriboside) (1,N(2)-gamma-hydroxypropano deoxyguanosine, gamma-HOPdG) is a major DNA adduct that forms as a result of exposure to acrolein, an environmental pollutant and a product of endogenous lipid peroxidation. gamma-HOPdG has been shown previously not to be a miscoding lesion when replicated in Escherichia coli. In contrast to those prokaryotic studies, in vivo replication and mutagenesis assays in COS-7 cells using single stranded DNA containing a specific gamma-HOPdG adduct, revealed that the gamma-HOPdG adduct was significantly mutagenic. Analyses revealed both transversion and transition types of mutations at an overall mutagenic frequency of 7.4 x 10(-2)/translesion synthesis. In vitro gamma-HOPdG strongly blocks DNA synthesis by two major polymerases, pol delta and pol epsilon. Replicative blockage of pol delta by gamma-HOPdG could be diminished by the addition of proliferating cell nuclear antigen, leading to highly mutagenic translesion bypass across this adduct. The differential functioning and processing capacities of the mammalian polymerases may be responsible for the higher mutation frequencies observed in this study when compared with the accurate and efficient nonmutagenic bypass observed in the bacterial system.     

Escherichia coli mutator (Delta)polA is defective in base mismatch correction: the nature of in vivo DNA replication errors

We constructed a set of Escherichia coli strains containing deletions in genes encoding three SOS polymerases, and defective in MutS and DNA polymerase I (PolI) mismatch repair, and estimated the rate and specificity of spontaneous endogenous tonB(+)-->tonB- mutations. The rate and specificity of mutations in strains proficient or deficient in three SOS polymerases was compared and found that there was no contribution of SOS polymerases to the chromosomal tonB mutations. MutS-deficient strains displayed elevated spontaneous mutation rates, consisting of dominantly minus frameshifts and transitions. Minus frameshifts are dominated by warm spots at run-bases. Among 57 transitions (both G:C-->A:T and A:T-->G:C), 35 occurred at two hotspot sites. PolI-deficient strains possessed an increased rate of deletions and frameshifts, because of a deficiency in postreplicative deletion and frameshift mismatch corrections. Frameshifts in PolI-deficient strains occurred within the entire tonB gene at non-run and run sequences. MutS and PolI double deficiency indicated a synergistic increase in the rate of deletions, frameshifts and transitions. In this case, mutS-specific hotspots for frameshifts and transitions disappeared. The results suggested that, unlike the case previously known pertaining to postreplicative MutS mismatch repair for frameshifts and transitions and PolI mismatch repair for frameshifts and deletions, PolI can recognize and correct transition mismatches. Possible mechanisms for distinct MutS and PolI mismatch repair are discussed. A strain containing deficiencies in three SOS polymerases, MutS mismatch repair and PolI mismatch repair was also constructed. The spectrum of spontaneous mutations in this strain is considered to represent the spectrum of in vivo DNA polymerase III replication errors. The mutation rate of this strain was 219x10(-8), about a 100-fold increase relative to the wild-type strain. Uncorrected polymerase III replication errors were predominantly frameshifts and base substitutions followed by deletions.     

Formation and genotoxicity of a guanine-cytosine intrastrand cross-link lesion in vivo

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/γ-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine–cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to γ-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 10⁹ nucleosides per Gy, which was ~300 times lower than that of 5-formyl-2′-deoxyuridine (0.011 lesions per 10⁶ nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G→T and a 1.2% G→C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo.     

Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid

Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts.

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.