Mutagenic DNA repair in Escherichia coli. VII. Constitutive and inducible manifestations.

Incubation of E. coli WP2 in the presence of chloramphenicol (CAP) for 90 min before and 60 min after γ-irradiation had no effect on the induction of Trp⁺ mutations. Bacteria that had been treated with CAP for 90 min prior to UV irradiation showed normal or near normal yields of induced mutations to streptomycin or colicin E2 resistance. Most of these mutations lost their photoreversibility (indicating “fixation”) during continued incubation with CAP for a further 60 min after irradiation, during which time neither protein nor DNA synthesis was detectable. It is suggested that CAP-sensitive protein synthesis is not required for mutagenic (error-prone) repair of lesions in pre-existing DNA, arguing against an inducible component in this repair.In contrast the frequency of UV-induced mutations to Trp⁺ (largely at suppressor loci) was drastically reduced by CAP pretreatment, confirming the need for an active replication fork for UV-mutagenesis at these loci. It is known from the work of others that CAP given after UV abolishes mutagenesis at these loci. We conclude that CAP-sensitive protein synthesis (consistent with a requirement for an inducible function) is necessary for mutagenic repair only in newly-replicated DNA (presumably at daughter strand gaps) and not in pre-existing DNA. The data are consistent with but do not prove the hypothesis that CAP-sensitive and insensitive modes of mutagenesis reflect minor differences in the operation of a single basic mutagenic repair system.     

Mutagenic repair in Escherichia coli. VIII. Effect of gyrB mutations on ultraviolet light mutagenesis

Introduction into Escherichia coli WP2 bacteria of a mutation in the gyrB locus previously shown to reduce the degree of chromosomal superhelicity caused a small decrease in the frequency of UV-induced mutations to streptomycin resistance (but not significantly) and to tryptophan independence (mostly ochre suppressors) in excision repair-proficient bacteria. It did not influence the 'broth effect' or the rate or extent of 'mutation frequency decline' of suppressor mutations. In an excision-deficient (uvrA 155) background the yield of UV-induced streptomycin-resistant mutations was lower in gyrB bacteria at all doses; the yield of tryptophan-independent mutations was slightly lower at low doses and slightly higher at high doses. In both excision-proficient and -deficient bacteria the yield of UV-induced mutations to rifampicin resistance was apparently lower in gyrB mutants but this could be due at least in part to a hypersensitivity of some Rifr gyrB bacteria to UV. The number of spontaneous tryptophan-independent mutations was lower in gyrB bacteria but this was almost certainly due to their poorer viability on tryptophan-limiting plates and not to a lower spontaneous mutation rate. In a temperature-sensitive presumed gyrase-deficient strain a small decrease in mutant yield at low doses was observed following incubation at restrictive temperature before UV. This was ascribed to an enhancement of excision repair. Our failure to find any significant effect of gyrB mutations does not support the hypothesis that hairpin formation (which should be dependent on a high degree of superhelicity) is involved in determining the 'broth effect', 'mutation frequency decline' or the probability that a mutation will occur spontaneously. Dramatic effects of superhelicity on UV mutagenesis also seem to be unlikely.     

DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli.

DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' leads to 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair.     

Mutagenic DNA repair in Escherichia coli. XXI. A stable SOS-inducing signal persisting after excision repair of ultraviolet damage.

Mutations to streptomycin resistance induced by ultraviolet light in Escherichia coli can lose their susceptibility to photoreversing light during excision repair and in the absence of chromosomal replication and protein synthesis, i.e., under conditions where SOS induction cannot occur. Using fusions of lac with sulA and umuC we have shown that after excision of UV damage in the presence of chloramphenicol there is a persisting, relatively stable signal capable of inducing SOS genes when protein sysnthesis is subsequently permitted. The persisting signal is formed roughly in proportion to the square of the UV dose and is about 30% photoreversible. It is suggested that the persisting SOS-inducing signal comprises a UV photoproduct (the target lesion) opposite a gap in the opposing DNA strand, and is formed by excision of one (the ancillary lesion) of a pair of closely opposed photoproducts. Calculations suggest that as few as two or three such configurations in a cell can lead to induction a sulA when protein synthesis is permitted. It is not clear whether these configurations can directly induce the SOS system because of their region of single-stranded DNA or whether the ultimate SOS-inducing signal is a more extensive single-stranded region formed when such configurations encounter a replication fork. Photoproduct/gap configurations have been previously suggested to be potentially mutagenic. UV-induced mutations to streptomycin resistance are mostly at A:T sites and are not photoreversible in fully SOS-induced bacteria in the absence of excision repair, indicating that they are not targeted at cyclobutane-type pyrimidine dimers. In SOS-induced excision-proficient bacteria there is about 39% photoreversibility which is rapidly lost after UV. This photoreversibility is attributed to many ancillary lesions being cyclobutane-type pyrimidine dimers which are excised leading to the exposure of target lesions on the opposing strand which, at these particular sites, are mostly non-photoreversible photoproducts.     

Mutagenic DNA repair in Escherichia coli. XIV. Influence of two DNA polymerase III mutator alleles on spontaneous and UV mutagenesis

Summary We introduced the dnaE486 and polC74 mutations (which are associated with decreased DNA polymerase III replication fidelity) into excision defective Escherichia coli strains with varying SOS responses. These mutations increased the UV-induced frequency of base pair substitution mutations in all strains tested, except recA430 and umuC122 derivatives. This UV mutator effect therefore requires expression of the SOS error-prone repair system. In recA441 lexA51 strains where the SOS system is constitutively expressed, the UV mutator effect of the dnaE alleles was similar in relative terms (though greater in absolute terms). Since these dnaE alleles decrease rather than increase survival after UV it is argued that they promote a burst of untargeted mutations close to UV photoproducts (hitch-hiking mutations) rather than increase the number of translesion synthesis events. The fact that there was no UV mutagenesis in dnaE486 umuC122 or polC74 umuC122 strains indicates that infidelity associated with these dnaE alleles did not of itself enable translesion synthesis to occur. The spontaneous mutator effect conferred by dnaE486 and polC74 was not affected by umuC122 or recA430 indicating that it is not dependent upon error-prone repair ability. In recA441 lexA51 bacteria, where SOS error-prone repair is constitutively induced, the mutator effect of dnaE486 was greater and was largely blocked by umuC122. It is suggested that spontaneously occurring cryptic lesions that are themselves unable to induce the SOS system are subject to translesion synthesis under these conditions and trigger a burst of hitch-hiking mutations that are therefore effectively umuC dependent.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

Degradation of Escherichia coli DNA synthesized after ultraviolet irradiation in the absence of repair

DNA degradation in Escherichia coli uvrA recA bacteria exposed to a low dose (0.07 J/m2) of ultraviolet radiation was studied. A considerable amount of the newly-synthesized DNA, which contains gaps opposite pyrimidine dimers, is broken down. In contrast, parental, dimer-containing DNA is resistant to radiation-induced degradation.     

Mutagenic DNA repair in Escherichia coli. XIII. Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

Mutagenic DNA repair in Escherichia coli XIII Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3′–5′-exonuclease activity of the ϵ proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect of UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the ϵ proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.     

DNA polymerase I modulates inducible stable DNA replication in Escherichia coli.

Mutants of Escherichia coli lacking RNase HI activity and cells induced for the SOS response express modes of DNA replication independent of protein synthesis, called constitutive and induced stable DNA replication, respectively. We report here that mutants deleted for the polA gene express induced stable DNA replication at approximately 25-fold the rate of wild-type cells, whereas constitutive stable DNA replication is not enhanced.     

Mutagenic DNA repair in Escherichia coli. XIX. On the roles of RecA protein in ultraviolet light mutagenesis

An experimental system was used in which His+ mutations induced by ultraviolet light (UV) arise from non-photo-reversible photoproducts whereas lethality is largely determined by photoreversible photoproducts. By exposing a strain with a deletion through recA to light immediately after UV, it was possible to examine mutagenesis under conditions where survival was not significantly different from 100%. No UV mutagenesis was seen in the absence of RecA protein even though the rest of the SOS system was fully expressed due to the presence of a defective LexA repressor and the active carboxy-terminal fragment of UmuD was present as a result of an engineered plasmid-borne gene. We conclude that RecA protein has a third essential function if UV mutagenesis is to be detected in excision-deficient-bacteria. Another experiment showed that in exerting this function RecA protein does not need activation by pyrimidine dimers elsewhere on the genome, in contrast to its protein-cleavage mediation functions with LexA and UmuD proteins. RecA1730 protein blocked UV mutagenesis unless delayed photoreversal was given showing that the third function of RecA protein is not in the misincorporation step. It is therefore most likely to be in the bypass step where UmuD' and UmuC are postulated to act, although the possibility cannot be excluded that RecA protein is required for some other survival function distinct from translesion synthesis.     

Mutagenic DNA repair in Escherichia coli. XVI. Mutagenesis by ultraviolet light plus delayed photoreversal in recA strains

Mutagenesis was demonstrable after delayed photoreversal of UV-irradiated strains carrying a recA deletion indicating that RecA protein is not essential for the misincorporation process that is revealed by delayed photoreversal. Moreover, the data suggest that RecA protein actually depresses misincorporation to varying extents depending on the recA allele. No delayed photoreversal was demonstrable in reA1 or recA56 bacteria unless the lexA102(ind-) allele was also present. It is suggested that the level of these RecA proteins may be lower in the lexA102(ind-) strains thus minimising their depressive effect. Delayed photoreversal mutagenesis in strains carrying the recA441 allele was not affected by either adenine or guanosine plus cytidine, substances which affect the proteolytic activity of RecA441 protein.     

Role of constitutive and inducible repair in radiation resistance of Escherichia coli

Radiation resistance of Escherichia coil cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recE, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB 1157 and highly radiation-resistant isogenic strain Gam(r)444. An optimal balance ensuring a high gamma resistance of the Gam(r)444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.