Binding and incision activities of UvrABC excinuclease on slipped DNA intermediates that generate frameshift mutations

Previous in vivo studies involving sequence 5'-CCCG1G2G3-3' (SmaI site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF) to any of the three guanine residues of the SmaI sequence induce, with different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro. In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the SmaI site (5'-CCCG1G2G3-3'). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the SmaI sequence in the bottom strand and were designated deltaC/3 and deltaG/3 when modified with the AAF on the third guanine residue in the top strand or deltaC/O and deltaG/O when unmodified. The modified homoduplex was designated SmaI/3. deltaC/O and deltaG/O were weakly recognized by UvrA2B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA2 from the UvrA2B-DNA complex occurred more readily in heteroduplexes than in the homoduplex. SmaI/3 and deltaC/3 were incised with equal efficiency, while deltaG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication.     

Site-specific frame-shift mutagenesis by the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-y1)-1-aminopyrene located in the (CG)3 sequence: effects of SOS, proofreading, and mismatch repair

1-Nitropyrene (1-NP), the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagen and tumorigen. Nitroreduction is a major pathway by which 1-NP is metabolized. Reductively activated 1-NP forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP), both in vitro and in vivo. In Salmonella typhimurium 1-NP induces a CpG deletion in a CGCGCGCG sequence. In Escherichia coli, however, mostly -1 and +1 frame-shifts are observed, which occur predominantly in 5'-CG, 5'-GC, and 5'-GG sequences. In order to determine the mechanism of mutagenesis by dGAP in a CpG repetitive sequence, we constructed a single-stranded M13 genome containing the adduct at the underscored deoxyguanosine of an inserted CGCGCG sequence. In E. coli strains with normal repair capability the adduct induced approximately 2% CpG deletions, which was 20-fold that of the control. With SOS, the frequency of frame-shift mutations increased to 2.6%, even though the frequency of CpG deletion accompanied 50% reduction. The enhancement in mutagenesis was due to a +1 frame-shift that occurred at a high frequency. In strains with a defect in methyl-directed mismatch repair, 50-70% increase in mutation frequency was observed. When these strains were SOS induced, frame-shift mutagenesis increased by approximately 100%. When transfections were carried out in dnaQ strains that are impaired in 3'-->5'exonuclease activity of DNA polymerase III, frame-shift mutagenesis increased 5-7-fold. dGAP-induced frame-shifts in the (CG)3 sequence, therefore, varied from 2% to 17% depending on the state of repair of the host cells. We conclude that dGAP induces both -2 and +1 frame-shifts in a CpG repetitive sequence and that these two mutagenic events are competing pathways. The CpG deletion does not require SOS functions, whereas the +1 frame-shifts are SOS-dependent. On the basis of the data in repair-deficient strains, it appears that both types of frame-shifts occurred as a result of misalignment, which are corrected primarily by the proofreading exonuclease of the DNA polymerase. Misaligned structures that escape the exonuclease are repaired by the methyl-directed mismatch repair, albeit with limited efficiency.     

Interaction of Escherichia coli DNA polymerase I (Klenow fragment) with primer-templates containing N-acetyl-2-aminofluorene or N-2-aminofluorene adducts in the active site

DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primer-templates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present.     

Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity

To better understand the mechanisms of SOS mutagenesis in the bacterium Escherichia coli, we have undertaken a genetic analysis of the SOS mutator activity. The SOS mutator activity results from constitutive expression of the SOS system in strains carrying a constitutively activated RecA protein (RecA730). We show that the SOS mutator activity is not enhanced in strains containing deficiencies in the uvrABC nucleotide excision-repair system or the xth and nfo base excision-repair systems. Further, recA730-induced errors are shown to be corrected by the MutHLS-dependent mismatch-repair system as efficiently as the corresponding errors in the rec+ background. These results suggest that the SOS mutator activity does not reflect mutagenesis at so-called cryptic lesions but instead represents an amplification of normally occurring DNA polymerase errors. Analysis of the base-pair-substitution mutations induced by recA730 in a mismatch repair-deficient background shows that both transition and transversion errors are amplified, although the effect is much larger for transversions than for transitions. Analysis of the mutator effect in various dnaE strains, including dnaE antimutators, as well as in proofreading-deficient dnaQ (mutD) strains suggests that in recA730 strains, two types of replication errors occur in parallel: (i) normal replication errors that are subject to both exonucleolytic proofreading and dnaE antimutator effects and (ii) recA730-specific errors that are not susceptible to either proofreading or dnaE antimutator effects. The combined data are consistent with a model suggesting that in recA730 cells error-prone replication complexes are assembled at sites where DNA polymerization is temporarily stalled, most likely when a normal polymerase insertion error has created a poorly extendable terminal mismatch. The modified complex forces extension of the mismatch largely at the exclusion of proofreading and polymerase dissociation pathways. SOS mutagenesis targeted at replication-blocking DNA lesions likely proceeds in the same manner.     

Solution structure of the aminofluorene-intercalated conformer of the syn [AF]-C8-dG adduct opposite a--2 deletion site in the NarI hot spot sequence context

This paper addresses structural issues related to the capacity of aminofluorene [AF] for frameshift mutations of the -2 type on C8 covalent adduct formation at the G3 site in the d(C-G1-G2-C-G3-C-C) NarI hot spot sequence. This problem has been approached from a combined NMR and relaxation matrix analysis computational structural study of the [AF]dG adduct in the d(C-G-G-C-[AF]G-C-C).d(G-G-C-C-G) sequence context at the 12/10-mer adduct level (designated [AF]dG.del(-2) 12/10-mer). The proton spectra of this system are of exceptional quality and are consistent with the formation of an AF-intercalated conformer with the modified guanine in a syn alignment displaced along with the 5'-flanking cytosine residue into the major groove. The solution structure has been determined by initially incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bound deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space and subsequently refined through restrainted molecular dynamics calculations based on a NOE distance and intensity refinement protocol. Strikingly, the [AF]dG.del(-2) 12/10-mer duplex adopts only one of two potential AF-intercalation alignments for the [AF]dG adduct opposite the -2 deletion site in the NarI sequence context with the extrusion of the dC-[AF]dG step favored completely over extrusion of the [AF]dG-dC step at the lesion site. This polarity establishes that the structural perturbation extends 5' rather than 3' to the [AF]dG lesion site in the adduct duplex. This structure of the [AF]dG adduct opposite a -2 deletion site shows distinct differences with conclusions reported on the alignment of the related acetylaminofluorene [AAF]dG adduct opposite a -2 deletion site in the identical NarI sequence context [Milhe, C., Fuchs, R. P. P., and Lefevre, J. F. (1996) Eur. J. Biochem. 235, 120-127]. In that study, qualitative NMR data without computational analysis were employed to conclude that the extrusion at the lesion site occurs at the [AAF]dG-dC step for the AAF-intercalated conformer of the adduct duplex. The structure of the [AF]dG adduct opposite a -2 deletion site determined in our group provides molecular insights into the architecture of extended slipped mutagenic intermediates involving aromatic amine intercalation and base-displaced syn modified guanines in AF and, by analogy, AAF-induced mutagenesis in the NarI hot spot sequence context.     

Lack of correlation between in vitro and in vivo replication of precisely defined benz-a-anthracene adducted DNAs

Like other polycyclic aromatic hydrocarbons, certain metabolites of benz[a]anthracene have been implicated as potent carcinogens. These effects are thought to be caused by the covalent binding of these species to nucleophilic groups on the bases of DNA. To address the molecular mechanisms by which these molecules induce mutations, this study employed oligonucleotides containing four site-specific N6 adenine-benz[a]anthracene diol epoxide adducts. Using a prokaryotic in vivo replication system, we have shown that both non-bay region anti-trans-benz[a]anthracene adducts are essentially nonmutagenic. In contrast, the bay region anti-trans-benz[a]anthracene lesions do induce point mutations at the adduct site. The mutagenic frequency of these bay region lesions is dependent on the stereochemistry about the adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated. The ability of the bacterial replication machinery to bypass the lesions does not correlate with the differences observed in their mutagenesis. While both non-bay region adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree. In vitro studies of the interactions of E. coli DNA polymerase III with these adducts have also been undertaken to further dissect the relationship between adduct structure and biological activity.     

The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme

The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.     

A slipped replication intermediate model is stabilized by the syn orientation of N-2-aminofluorene- and N-2-(acetyl)aminofluorene-modified guanine at a mutational hotspot

The Escherichia coli NarI restriction enzyme recognition site 5'G1G2C3G4C5C63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 4147-4151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G4 by the related aromatic amine 2-aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.     

Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein

In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal role in SOS-induced mutagenesis. This role can be separated genetically into three steps: (i) depression of the SOS regulon by mediating the posttranslational cleavage of the LexA repressor, (ii) activation of UmuD'-like proteins by mediating cleavage of the UmuD-like proteins, and (iii) a direct step, possibly to interact with and to target the Umu-like mutagenesis proteins to lesions in DNA. We have analyzed RecA's third role biochemically using protein affinity chromatography and an agarose-based DNA mobility-shift assay. RecA730 protein from a crude cell extract was specifically retained on UmuD and UmuD' protein affinity columns, suggesting that these proteins physically interact. Normally, neither UmuD nor UmuD' shows any affinity for DNA. In the presence of RecA protein, however, UmuD and UmuD' were targeted to DNA. RecA1730 protein, which is defective for UmuD' but proficient for MucA'-promoted mutagenesis, showed a dramatically reduced capacity to target UmuD' to DNA but was able to target a significant portion of MucA' to DNA. These data support the suggestion that the direct role of RecA protein in SOS-induced mutagenesis is to interact with and target the Umu-like mutagenesis proteins to DNA.     

Aromatic amine DNA adduct formation in chronically-exposed mice: considerations for human comparison

Lifetime chronic exposure of mice to the aromatic amines 4-aminobiphenyl (ABP) and 2-acetylaminofluorene (AAF) produces liver and urinary bladder tumors. In parallel experiments, DNA adduct levels in target tissues reach a steady-state (a balance between adduct formation and removal) after about four weeks of either AAF or ABP ingestion. For these and other carcinogens, steady-state DNA adduct levels most frequently increase linearly with dose, but the formation of tumors also depends upon a variety of factors, including the proliferative capacity of the target tissue, the sex of the animal, genotoxic properties of the specific adducts formed, and other unknown events. Chronic dosing experiments in animal models are of interest for human risk assessment because human exposure is typically intermittent, involving repeated exposures. However, it is to be expected that in a genetically-diverse human population, where the lifetime averages > 70 years, the relationship between tumorigenesis and DNA adduct formation will be relatively more complex than that observed in mice. From our studies of chronic ABP exposure in male mice, we have obtained the daily dose of ABP and the steady-state level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) adduct associated with a 50% mouse bladder tumor incidence. Our attempt at a human extrapolation for adducts and urinary bladder cancer in smoking males (20-40 cigarettes/day) is based on the ABP dose per cigarette, values for the dG-C8-ABP adduct in bladder biopsies of lifetime heavy smokers at age approximately 70, and the smoking-related bladder tumor incidence (absolute lifetime risk) for Caucasian males in the United States aged 65-84 years. The extrapolation has produced two major predictions, one related to adduct formation and the other related to tumorigenesis. First, the observed level of smoking-related dG-C8-ABP in DNA of human bladder epithelium, expressed as a function of daily ABP intake, is about 3500-times higher than similar data for mice, which suggests that humans may perform the biotransformation of ABP more efficiently than mice. Second, at a similar bladder tumor incidence, mouse bladder contained adduct concentrations that were much higher than those observed in human bladder; for example, at a 2.6% tumor incidence, mouse bladder contained an average of 55.5 fmol dG-C8-ABP/microgram DNA (1850 adducts/10(8) nucleotides), while bladders from Caucasian male smokers contained an average of 0.036 fmol dG-C8-ABP/microgram DNA (1.2 adducts/10(8) nucleotides). This suggests that factors other than ABP-DNA adducts, such as adducts of other carcinogens, the influence of promoters, and synergistic effects of all of these factors contribute substantially to smoking-related bladder cancer in humans.     

A molecular mechanics and dynamics study of the minor adduct between DNA and the carcinogen 2-(acetylamino)fluorene (dG-N2-AAF)

Experimental studies involving the carcinogenic aromatic amine 2-(acetylamino)fluorene (AAF) have afforded two acetylated DNA adducts, the major one bound to C8 of guanine and a minor adduct bound to N2 of guanine. The minor adduct may be important in carcinogenesis because it persists, while the major adduct is rapidly repaired. Primer extension studies of the minor adduct have indicated that it blocks DNA synthesis, with some bypass and misincorporation of adenine opposite the lesion [Shibutani, S., and Grollman, A.P. (1993) Chem. Res. Toxicol. 6, 819-824]. No experimental structural information is available for this adduct. Extensive minimized potential energy searches involving thousands of trials and molecular dynamics simulations were used to study the conformation of this adduct in three sequences: I, d(C1-G2-C3-[AAF]G4-C5-G6-C7).d(G8-C9-G10-C11-G12-C13-G14+ ++); II, the sequence of Shibutani and Grollman, d(C1-T2-A3-[AAF]G4-T5-C6-A7).d(T8-G9-A10-C11-T12-A13-G14); and III, which is the same as II but with a mismatched adenine in position 11, opposite the lesion. AAF was located in the minor groove in the low-energy structures of all sequences. In the lowest energy form of the C3-[AAF]G4-C5 sequence I, the fluorenyl rings point in the 3' direction along the modified strand and the acetyl in the 5' direction. These orientations are reversed in the second lowest energy structure of this sequence, and the energy of this structure is 1.4 kcal/mol higher. Watson Crick hydrogen bonding is intact in both structures. In the two lowest energy structures of the A3-[AAF]G4-T5 sequence II, the AAF is also located in the minor groove with Watson-Crick hydrogen bonding intact. However, in the lowest energy form, the fluorenyl rings point in the 5' direction and the acetyl in the 3' direction. The energy of the structure with opposite orientation is 5.1 kcal/mol higher. In sequence III with adenine mismatched to the modified guanine, the lowest energy form also had the fluorenyl rings oriented 5' in the minor groove with intact Watson-Crick base pairing. However, the mispaired adenine adopts a syn orientation with Hoogsteen pairing to the modified guanine. These results suggest that the orientation of the AAF in the minor groove may be DNA sequence dependent. Mobile aspects of favored structures derived from molecular dynamics simulations with explicit solvent and salt support the essentially undistorting nature of this lesion, which is in harmony with its persistence in mammalian systems.     

Repair of propanodeoxyguanosine by nucleotide excision repair in vivo and in vitro

Repair of the exocyclic DNA adduct propanodeoxyguanosine (PdG) was assessed in both in vivo and in vitro assays. PdG was site-specifically incorporated at position 6256 of M13MB102 DNA, and the adducted viral genome was electroporated into repair-proficient and repair-deficient Escherichia coli strains. Comparable frequencies of PdG --> T and PdG --> A mutations at position 6256 were detected following replication of the adducted genomes in wild-type E. coli strains. A 4-fold increase in the frequencies of transversions and transitions was observed in E. coli strains deficient in Uvr(A)BC-dependent nucleotide excision repair. A similar increase in the replication of the adduct containing strand was observed in the repair-deficient strains. No change in the frequency of targeted mutations was observed in strains deficient in one or both of the genes coding for 3-methyladenine glycosylase. Incubation of purified E. coli Uvr(A)BC proteins with a duplex 156-mer containing a single PdG adduct resulted in removal of a 12-base oligonucleotide containing the adduct. Incubation of the same adducted duplex with Chinese hamster ovary cell-free extracts also resulted in removal of the adduct. PdG was a better substrate for repair by the mammalian nucleotide excision repair complex than the bacterial repair complex and was approximately equal to a thymine-thymine dimer as a substrate for the former. The results of these in vivo and in vitro experiments indicate that PdG, a homolog of several endogenously produced DNA adducts, is repaired by the nucleotide excision repair pathway.     

DNA nick processing by exonuclease and polymerase activities of bacteriophage T4 DNA polymerase accounts for acridine-induced mutation specificities in T4

Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage.     

A comparison of the detection sensitivity of lymphocyte membrane antigens using fluorescein and phosphor immunoconjugates

In Escherichia coli, UV and many chemicals appear to cause mutagenesis by a process of translesion synthesis that requires some form of DNA polymerase III and the SOS-regulated proteins UmuD, UmuC and RecA. An analysis of SOS mutagenesis offers insights into the molecular basis of induced mutagenesis and into mechanisms of DNA damage tolerance.     

Anti-mutagenic activity of DNA damage-binding proteins mediated by direct inhibition of translesion replication

DNA lesions that block replication can be bypassed in Escherichia coli by a special DNA synthesis process termed translesion replication. This process is mutagenic due to the miscoding nature of the DNA lesions. We report that the repair enzyme formamido-pyrimidine DNA glycosylase and the general DNA damage recognition protein UvrA each inhibit specifically translesion replication through an abasic site analog by purified DNA polymerases I and II, and DNA polymerase III (alpha subunit) from E. coli. In vivo experiments suggest that a similar inhibitory mechanism prevents at least 70% of the mutations caused by ultraviolet light DNA lesions in E. coli. These results suggest that DNA damage-binding proteins regulate mutagenesis by a novel mechanism that involves direct inhibition of translesion replication. This mechanism provides anti-mutagenic defense against DNA lesions that have escaped DNA repair.     

Overexpression of DNA polymerase beta in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs

DNA polymerase beta (pol beta) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol beta cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol beta. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol beta overexpression in cancer predisposition and tumor progression during chemotherapy.     

An ethanolic-aqueous extract of Curcuma longa decreases the susceptibility of liver microsomes and mitochondria to lipid peroxidation in atherosclerotic rabbits

Escherichia coli K12 assay-system is designed in order to detect bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication. The assay is composed of four tests aimed at the detection of inhibition of spontaneous and induced mutations (Tests A and B) and at the estimation whether the anti-mutagenic agent acts by increasing the fidelity of DNA replication (Test B), by inhibition of SOS error prone repair (Test C), or by favoring error-free recombinational repair (Test D). In Test A, repair proficient strain and its uvrA counterpart are used for detection of spontaneous and UV-induced mutations, while in Test B mismatch repair deficient strains (mutH, mutS, mutL and uvrD) are used for amplified detection of spontaneous mutations caused by replication errors. In Test C, repair proficient strain carrying sfiA::lacZ fusion is used for measuring the level of SOS induction by monitoring the level of beta-galactosidase. In Test D, the strains carrying different recA alleles (recA+, recA730 and DeltarecA) are used for measuring intrachromosomal recombination between nonoverlapping deletions in duplicated lac operon, by monitoring Lac+ recombinants. The assay-system is validated with model bioantimutagens and used for detection of anti-mutagenic potential of different terpenoid fractions from sage (Salvia officinalis L.). Extract E1/3 of cultivated sage, distinguished from others by its high content of monoterpenoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Tests B and C. In Test D, it enhances intrachromosomal recombination in untreated and UV-irradiated recA+ and recA730 strains. The results suggest that the protective effect is due to stimulation of recombinational repair, similarly to coumarin. We speculate that monoterpenoids from sage enhance genetic recombination by intervening in a formation of RecA-DNA complex and channeling it into recombination reaction.     

Mutagenicity and genotoxicity of the major DNA adduct of the antitumor drug cis-diamminedichloroplatinum(II)

The mutagenicity and genotoxicity of cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] (G*G*), the major DNA adduct of the antitumor drug cisplatin, has been investigated in Escherichia coli. A duplex bacteriophage M13 genome was constructed to contain the G*G* adduct at a specific site in the (-) strand. The singly platinated duplex genome exhibited a survival of 22% relative to that of the unplatinated control genomes, and this value rose to 38% in cells treated with ultraviolet light to induce the SOS response. Singly platinated single-stranded genomes were also produced. Replication of the single- and double-stranded genomes in vivo yielded SOS-dependent, targeted mutations at frequencies of 1.3% and 0.16%, respectively. The mutagenic specificity of G*G* in both single- and double-stranded DNA was striking in that 80-90% of the mutations occurred at the 5'-platinated G. Approximately 80% of the mutations were G-->T transversions at that site. A model of mutagenesis is presented to explain this mutational specificity with respect to current understanding of platinum-DNA adduct structure.     

C1 inhibitor gene sequence facilitates frameshift mutations

Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema. Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations. To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains. These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency. Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored. Both pairs of isogenic strains had reversion frequencies of approximately 10(-8). These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations. Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis. This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication. Models explaining the slippage can be drawn using the lagging strand of the replication fork. In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.