uvrA Cells Research Articles

Frameshift Mutations Produced by 9-Aminoacridine in Wild-Type, uvrA and recA Strains of Escherichia coli; Specificity Within a Hotspot

The endogenous tonB gene of Escherichia coli was used as a target for 9-aminoacridine-induced mutations that were identified in recA(-) and uvrA(-) cells. The cytotoxicity of 9-aminoacridine was enhanced in the uvrA and recA strains compared to the wild-type strain, and the mutagenicity of 9-aminoacridine in the uvrA and recA strains was similar to that in the wild type. For all three strains, the most common mutations were minus frameshifts in repetitive G:C base-pairs followed by minus frameshifts in nonrepetitive G:C base-pairs. 9-aminoacridine-induced minus frameshifts in the wild-type strain were distributed with several hot and warm spots. These sites were also hot and warm spots for minus frameshifts in the recA and uvrA stains. Furthermore, they were hot and warm sites in a 9-aminoacridine-treated strain carrying the target tonB gene oriented in the opposite direction. 9-Aminoacridine is known to interact with DNA to form intercalations which are involved in minus frameshift mutagenesis. In this study, we therefore argue that 1) 9-aminoacridine can induce bulky DNA lesions which are excised by nucleotide excision repair and not involved in mutagenesis, 2) the presence or absence of a recA-dependent repair pathway does not influence the mutagenic effect of 9-aminoacridine, and 3) both leading strand and lagging strand replication equally produce minus frameshifts, therefore gene orientation is not an important determinant of the formation of hot and warm spots by 9-aminoacridine.

Mutagenicity and co-mutagenicity of static magnetic fields detected by bacterial mutation assay

Possible mutagenic and co-mutagenic effects of strong static magnetic fields were estimated using bacterial mutagenicity test. Mutagenic potential of static magnetic fields up to 5T (T:1T=10,000 G) was not detected by the bacterial mutagenicity test using four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA either in the pre-incubation method or in the plate incorporation method. In the co-mutagenicity test, E. coli WP2 uvrA cells were treated with various chemical mutagens and were simultaneously exposed to a 2T or a 5T static magnetic field. Mutation rate in the exposed group was significantly higher than that in the non-exposed group when cells were treated with N-ethyl- N′-nitro- N-nitrosoguanidine (ENNG), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), 4-nitroquinoline- N-oxide (4-NQO), 2-amino-3-methyl-3 H-imidazo[4,5-f]quinoline (IQ) or 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2). The mutagenicity of 2-aminoanthracene (2-AA), 9-aminoacridine (9-AA), N 4-aminocytidine and 2-acetoamidofluorene (2-AAF) was not affected by the magnetic field exposure. Possible mechanisms of the co-mutagenicity of magnetic fields are discussed.

Role of E. coli DNA helicase II in a umuCD-dependent Wiegle reactivation

Wiegle reactivation is a manifestation of a umuCD-mediated enhancement in the replication of damaged phage DNA (Caillet-Fauquet et al., 1977; Defais et al., 1989; Rajagopalan et al., 1992). I have obtained Wiegle reactivation of lambda by irradiating the uvrA cells in rich growth medium. This Wiegle reactivation was lost upon transduction of the umuC mutation into the strain. There was also a drastic reduction of Wiegle reactivation in an isogenic strain carrying the uvrA mutation and deletion of the uvrD gene. ( uvrD codes for E. coli DNA helicase II). The effect of the uvrD deletion on Wiegle reactivation can indicate a specific requirement of DNA helicase II for the unwinding of damaged phage DNA during its replication or (and) an inefficient induction of the SOS response in Δ uvrD uvrA cells.

Targeted A --> T and G --> T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol epoxide of dibenz[a,j]anthracene in M13mp7L2.

The studies described in this report directly examined the mutagenicity in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguanosine (dGuo) adduct derived from (+)-anti-dibenz[a,j]-anthracene-3,4-diol 1,2-epoxide [(+)anti-DB[a,j]A-DE] that were site-specifically placed in a single-stranded M13mp7L2 replication vector. An 11-base oligonucleotide (5'-CTC ACG CTT CT-3') containing either a single (+)anti-DB[a,j]A-DE--trans-N2-dGuo or (+)anti-DB[a,j]A-DE--trans-N6dAdo adduct was successfully incorporated into single-stranded M13mp7L2 plasmid via ligation. In vitro studies using E. coli DNA polymerase I (Klenow fragment)indicated that both adducts were effective blocks for polymerase action. E. coli strains JM103 and JM103 uvrA6 were subsequently transformed with control (unadducted) and adduct-containing M13mp7L2 constructs followed by analysis of progeny DNA. In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors. Activation of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacterial strain transformed with adduct-containing vectors. Targeted mutations were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors. In JM103 cells, (+)anti-DB[a,j]A-DE--N6-dAdo induced exclusively A --> t transversions and (+)anti-DB[a,j]A-DE--N2-dGuo induced exclusively G --> T transversions. In JM103 uvrA6 cells, similar targeted transversion mutations were also obtained except that a few C deletions (i.e., aprroximately 10% of the mutations) were detected immediately 3' to the dAdo adduct. While mutagenesis was SOS dependent in JM103 cells [<0.15% (-SOS) vs approximately 1.3% (+SOS)], it appeared to be SOS independent in JM103 uvrA6 cells (approximately 1-2% in the presence or absence of SOS induction). It is argued that adduct-induced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms. In contrast, A --> T mutations are unlikely to arise via a misinformational pathway, which provides the strongest support to date that bulky DNA adducts can induce mutations via a noninformational pathway.

Involvement of RecF pathway recombination genes in postreplication repair in UV-irradiated Escherichia coli cells

Mutations affecting the RecF pathway of recombination (recF, recG, recJ, recN, recO, recQ, recR, ruvA, ruvC) were systematically introduced into two sets of strains: (a) uvrA and uvrA recA2020, (b) uvrA recBC sbcBC and uvrA recBC sbcBC recA2020. We examined: (i) the effect of these mutations on the repair of DNA daughter-strand gaps which are produced in the nascent DNA synthesized after UV irradiation, and (ii) the ability of recA2020 (a suppressor for the recF mutation) to suppress the UV radiation sensitivity caused by these mutations. In the uvrA cells, mutations in recF, recR or recO genes produced a major deficiency in the repair of daughter-strand gaps, whereas mutations in recJ, recG, recN, recQ, ruvA or ruvC genes had no effect on the repair of daughter-strand gaps. In both uvrA and uvrA recBC sbcBC backgrounds, the UV radiation sensitivity caused by recF, recG, recR, recO, ruvA, or ruvC mutation was partially suppressed by recA2020, whereas the UV radiation sensitivity caused by recJ, recN, or recQ mutations was not suppressed by recA2020. Partial suppression of the UV sensitivity of recG, ruvA and ruvC mutants was not observed with other suppressors for recF, i.e., recA441, recA720 and recA730. Taken together, these results further support the notion that the recF, recR and recO gene products (abbreviated as RecFOR) function at the same step in recombination repair, possible as a complex. It also suggests that this putative RecFOR) complex does not contain proteins encoded by other genes involved in the RecF pathway of recombination.

The role of formaldehyde and S-chloromethylglutathione in the bacterial mutagenicity of methylene chloride

Methylene chloride was less mutagenic in Salmonella typhimurium TA100/NG-11 (glutathione-deficient) compared to TA100, indicating that glutathione is involved in the activation of methylene chloride to a mutagen in bacteria. In rodents, the pathway of methylene chloride metabolism utilizing glutathione produces formaldehyde via a postulated S-chloromethylglutathione conjugate (GSCH 2Cl). Formaldehyde is known to cause DNA-protein cross-links, and GSCH 2Cl may act as a monofunctional DNA alkylator by analogy with the glutathione conjugates of 1,2-dihaloalkanes. The lack of sensitivity of Salmonella TA100 towards formaldehyde (Schmid et al., Mutagenesis, 1 (1986) No. 6, 427–431) suggests that GSCH 2Cl is responsible for methylene chloride mutagenicity in Salmonella. In Escherichia coli K12 (AB1157), formaldehyde was mutagenic only in the wild-type, a characteristic shared with cross-linking agents, whereas 1,2-dibromoethane (1,2-DBE) was more mutagenic in uvrA cells (AB1886). Methylene chloride, activated by S9 from mouse liver, was mutagenic only in wild-type cells, suggesting a mutagenic role for metabolically derived formaldehyde in E. coli. Mouse-liver S9 also enhanced the cell-killing effect of methylene chloride in the uvrA, and a recA/uvrA double mutant (AB2480) which is very sensitive to DNA damage. This pattern was consistent with formaldehyde damage. However, a mutagenic role in bacteria for the glutathione conjugate of methylene chloride cannot be ruled out by these E. coli experiments because S9 fractions did not increase 1,2-DBE mutagenicity, suggesting lack of cell wall penetration by this reactive species. Rat-liver S9 did not activate methylene chloride to a bacterial mutagen or enhance methylene chloride-induced cell-killing, which is consistent with the carcinogenicity difference between the species.

Cosuppression of recF, recR and recO mutations by mutant recA alleles in Escherichia coli cells

The ability of recA718, recA720, recA730, recA750 and two known recA(Srf) alleles (recA2020 and recA441) to act as suppressors of recF, recR and recO mutations was examined by studying their UV radiation sensitivity in uvrA cells of Escherichia coli. With the exception of recA718, all the mutant recA alleles examined were able to suppress the UV radiation sensitivity caused by recF, recR or recO mutations, but not by the recB mutation. The suppression by recA750 was minimal. The suppression of recF, recR and recO mutations by other recA alleles was more pronounced, but none of them could exert full suppression. Heterozygotes containing a mutant recA allele (recA720, recA730 or recA441) and recA2020 (or recA803, another known recA(Srf) allele) failed to produce any additive or synergistic suppression of recF, indicating that these suppressors did not use different mechanisms for their suppression. Similar to a requirement of recJ+ in the suppression of recF, the suppession of recR and recO mutations by mutant recA alleles also required recJ+. The similar phenotypes conferred by recF, recR, and recO mutations and the observations that a number of mutant recA alleles can cosuppress these three mutations suggest that the recF, recR and recO gene products may function together as a complex.

Similar-sized daughter-strand gaps are produced in the leading and lagging strands of DNA in UV-irradiated E. coli uvrA cells

The nascent DNA synthesized after UV irradiation contained discontinuity, i.e., daughter-strand gaps. The sizes of these gaps produced in the leading and lagging strands of UV-irradiated Escherichia coli cells were determined by using strand-specific DNA probes. The DNA isolated from irradiated uvrA delta(lac-pro) cells was hybridized with the 32P-labeled single-stranded DNA probes. After digestion with S1 nuclease, the sizes of the bound radioactive DNA fragments were determined by electrophoresis in an alkaline agarose gel. It was found that the average size of gaps produced in the leading strand was about 0.12 kb, whereas those produced in the lagging strand was slightly smaller than 0.12 kb. No gaps larger than 0.5 kb were detected.

Recombinagenic processing of UV-light photoproducts in nonreplicating phage DNA by the Escherichia coli methyl-directed mismatch repair system.

Nonreplicating lambda phage DNA in homoimmune Escherichia coli lysogens provides a useful model system for study of processes that activate DNA for homologous recombination. We measured recombination by extracting phage DNA from infected cells, using it to transfect recA recipient cells, and scoring the frequency of recombinant infective centers. With unirradiated phage, recombinant frequencies were less than 0.1%. However, recombination could be increased over 300-fold by prior UV irradiation of the phages. The dependence of recombination on UvrA function varied greatly with UV dose. With phage irradiated to 20 J/m2, recombinant frequencies in repressed infections of uvr+ bacteria were one-fifth those in uvrA infections; with phages irradiated to 100 J/m2, frequencies in uvr+ infections were thirty times higher than in uvrA infections. Most UV-stimulated recombination in uvrA infections appeared to depend on the bacterial methyl-directed mismatch-repair system: frequencies were depressed 5-20-fold in uvrA bacteria also lacking MutH, MutL or MutS functions, and recombinant frequencies decreased with increasing GATC-adenine methylation of phage stocks. The biological activity of nonreplicating UV-irradiated phage DNA declined with time after infection of uvrA cells; this decline was photoproduct-dependent, more marked for undermethylated than overmethylated phage DNA, and depended on host MutHLS functions. In uvr+ bacteria, where the UvrABC system provided an alternative, apparently less efficient, route to recombinagenic DNA, UV-stimulated recombinant frequencies were about twice as high in mutH or mutLS as in mut+ cells, in agreement with hyper-rec mut effects previously described by others.

DNA sequence changes in mutations induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr + and uvrA cells

Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E. coli uvr+ and excision-deficient uvrA cells. Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr+ cells. This conclusion is confirmed by analysis of published results for genes in both uvr+ and uvr- cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E. coli lacI gene and of the lambda phage cI repressor gene. Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E. coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair. Otherwise, mutagenesis in hamster cells appeared similar to that in E. coli uvr+ cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells. Phenotypes of 6-thioguanine-resistant E. coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria.

The thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3' thymine-to-cytosine transitions in Escherichia coli.

We have constructed single-stranded, M13-based vectors that contain a specifically located thymine-thymine pyrimidine-pyrimidone(6-4) UV photoproduct and have used these to estimate the frequency and accuracy of DNA replication past this adduct in uvrA6 cells of Escherichia coli. Both the normal and the Dewar valence photoisomer of the (6-4) adduct were studied. In the absence of SOS induction, vectors carrying the photoproducts were rarely replicated; relative to the lesion-free control, 1.9% of vectors carrying the normal (6-4) isomer produced plaques, and with the Dewar valence isomer the proportion was 0.4%. In SOS-induced cells, these frequencies rose to 22.1% and 12.3%, respectively. The error frequency of replication past the normal isomer in SOS-induced cells was high; in a random sample of 185 progeny phage analyzed, 169 (91%) contained mutations, all of which were targeted. Equally striking, a high proportion of the mutations (158/169; 93%) were of only one type, namely 3' T----C transitions. Both the error frequency and the specificity were much reduced with the Dewar valence isomer; overall, 74/140 (53%) of the phage analyzed were mutant, and of these only 34 (46%) entailed the 3' T----C transition. We speculate that the high error frequency and specificity arise from the formation of a stable T-G base pair, involving hydrogen bonds at O-2 and N-3 in the pyrimidone ring. Potential hydrogen bonds at these sites are coplanar in the normal but not in the Dewar isomer, perhaps explaining the reduced specificity of mutagenesis with the latter adduct.

Comparative survival of aminobiphenyl- and aminofluorene- substituted plasmid DNA in Escherichia coli Uvr endonuclease deficient strains

pBR322 plasmid DNA, randomly substituted with arylamine moieties, was introduced into Escherichia coli Uvr endonuclease deficient strains. Plasmid survival was determined by selection in the presence of ampicillin. Modification of plasmid DNA with N-acetoxy-N-trifluoroacetylaminobiphenyl yielded primarily N-(deoxyguanosin-8-yl)-4-aminobiphenyl residues. Reaction of DNA with N-acetoxy-N-acetylaminobiphenyl produced only N-(deoxyguanosin-8-yl)-4-acetylaminobiphenyl adducts. The aminobiphenyl (ABP) and acetylaminobiphenyl adducts reduced the ability of the plasmid DNA to transform E. coli to approximately the same extent in a wild-type strain. In uvrA, uvrB and uvrC, i.e. Uvr endonuclease deficient strains, both adducts produced equivalent decreases in survival, however, the reduction in survival was much more pronounced in the uvr- cells than in the wild-type strain. A similar pattern of toxicity was observed with plasmids carrying N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adducts, although the acetylaminofluorene adduct was approximately 5-fold more effective in reducing the biological activity of the plasmid. In contrast, the deacetylated aminofluorene (AF) lesion, N-(deoxyguanosin-8-yl-2-aminofluorene, exhibited relatively little effect on plasmid survival in uvrA and uvrB cells as compared to the wild-type strain, even though the survival of both ABP and AF adducts was essentially similar in the uvrC and wild-type strains. These data demonstrate that (i) both the deacetylated and acetylated lesions are subject to repair by the Uvr endonuclease complex, and (ii) the presence of the N-acetyl group is not the sole determinant of the differential effects of arylamine adducts in uvr cells. These observations provide indirect evidence that both the N-acetyl and aryl moieties of these adducts alter the conformation of DNA.

In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl- or O6-n-butyl-guanine in a T:G base pair.

We have previously reported some effects of DNA repair on the transition frequencies produced by an O6-methyl-guanine (MeG) or an O6-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage phi X174 form I' DNA (Chambers et al. 1985). We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site. The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA). The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype. The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E. coli AB1886). A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884). These data, like our previous data, indicate BuG is repaired primarily by excision. In contrast to this, the transition frequency from T:MeG is high (5 +/- 2%) in cells with normal repair. After induction of alkyl transfer repair in E. coli AB1157, the transition frequency goes up 5-fold. Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E. coli AB2463). The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of DNA polymerase I in postreplication repair: a reexamination with Escherichia coli delta polA

Using strains of Escherichia coli K-12 that are deleted for the polA gene, we have reexamined the role of DNA polymerase I (encoded by polA) in postreplication repair after UV irradiation. The polA deletion (in contrast to the polA1 mutation) made uvrA cells very sensitive to UV radiation; the UV radiation sensitivity of a uvrA delta polA strain was about the same as that of a uvrA recF strain, a strain known to be grossly deficient in postreplication repair. The delta polA mutation interacted synergistically with a recF mutation in UV radiation sensitization, suggesting that the polA gene functions in pathways of postreplication repair that are largely independent of the recF gene. When compared to a uvrA strain, a uvrA delta polA strain was deficient in the repair of DNA daughter strand gaps, but not as deficient as a uvrA recF strain. Introduction of the delta polA mutation into uvrA recF cells made them deficient in the repair of DNA double-strand breaks after UV irradiation. The UV radiation sensitivity of a uvrA polA546(Ts) strain (defective in the 5'----3' exonuclease of DNA polymerase I) determined at the restrictive temperature was very close to that of a uvrA delta polA strain. These results suggest a major role for the 5'----3' exonuclease activity of DNA polymerase I in postreplication repair, in the repair of both DNA daughter strand gaps and double-strand breaks.

Comparison of the rep-38 and mmrA1 mutations of Escherichia coli

The rep-38 and mmrA1 mutations are located very close to each other (∼85 min), and have been suggested to be allelic. To address this question we have compared the phenotypes of the mmrA1 and rep-38 mutants. Both the mmrA1 and rep-38 mutations blocked the enhanced killing and inhibition of postreplication repair by rich growth medium that occurs in UV-irradiated Escherichia coli K-12 uvrA cells, i.e., the mmrA1 and rep-38 strains did not show minimal medium recovery (MMR). However, ΦX174 bacteriophage propagated well in mmrA1 strains, but not in rep-38 strains; a rep mutation sensitized a uvrA strain to UV irradiation, but a mmrA mutation did not. During chloramphenicol treatment, the rep-38 strain showed a larger amount of residual DNA synthesis than observed in the mmrA1 strain. The mmrA1 mutation appears to be a dominant mutation. This was determined by the failure of either plasmid pLC44-7 or episome F′KLF11, both of which carry the mmrA + gene, to complement the Mmr − phenotype of a uvrA mmrA strain. Plasmid pLC44-7 is known to complement the rep-38 mutation, suggesting that rep-38 is a recessive mutation. Although certain of the phenotypes of the rep and mmrA mutants are similar, a number are quite different. These differences suggest that these two mutations are not allelic.

Amplified RNase H activity in Escherichia coli B/r increases sensitivity to ultraviolet radiation.

Strains of E. coli B/r transformed with the plasmid pSK760 were found to be sensitized to inactivation by ultraviolet radiation (UV) and to have elevated levels of RNase H activity. Strains transformed with the carrier vector pBR322 or the plasmid pSK762C derived from pSK760 but with an inactivated rnh gene were not sensitized. UV-inactivation data for strains having known defects in DNA repair and transformed with pSK760 suggested an interference by RNase H of postreplication repair: uvrA cells were strongly sensitized, wild-type and uvrA recF cells were moderately sensitized and recA cells were not sensitized; and minimal medium recovery was no longer apparent in sensitized uvrA cells. Biochemical studies showed that post-UV DNA synthesis was sensitized and that the smaller amounts of DNA synthesized after irradiation, while of normal reduced size as indicated by sedimentation position in alkaline sucrose gradients, did not shift to a larger size (more rapidly sedimenting) upon additional incubation. We suggest an excess level of RNase H interferes with reinitiation of DNA synthesis on damaged templates to disturb the normal pattern of daughter strand gaps and thereby to inhibit postreplication repair.

The relative cytotoxicity and mutagenicity of cyclobutane pyrimidine dimers and 〈6—4〉 photoproducts in escherichia coli cells

In order to calculate the relative cytotoxicity and mutagenicity of 〈5—6〉 cyclobutane pyramidine dimers and 〈6—4〉 photoproducts, we have measured survival and mutation induction in UV-irradiated excision-deficient deficient E. coli uvrA cells, with or without complete photoreactivation of the 〈5–6〉 dimers. Radioimmunoassays with specificity for 〈5–6〉 dimers or 〈6-4〉 photoproducts have shown that maximum photoreactivation eliminates all of the 〈5–6〉 dimers produced up to 10 Jm −2 254-nm light, while it has no effect on 〈6-4〉 photoproducts. These results were confirmed by measuring the frequency of T4 endonuclease V-sensitive sites. Based on the based fit equations for survival and mutation induction, we have found that the calculated cytotoxicity of 〈6-4〉 photoproducts is similar to that of 〈5–6〉 dimers; however, the former is much more mutagenic than the latter.

A mechanism for rich-medium inhibition of the repair of daughter-strand gaps in the deoxyribonucleic acid of UV-irradiated Escherichia coli K12 uvrA

Ultraviolet-irradiated Escherichia coli K12 uvrA(B,C) cells show higher survival if plated on minimal growth medium (MM) rather than on rich growth medium (RM). This phenomenon has been referred to as ‘minimal medium recovery’ (MMR). UV-irradiated (4 J/m 2) uvrA cells showed a similar rate of protein synthesis, whether incubated in MM or RM, however, they showed a severe depression in DNA synthesis when incubated in MM that lasted for about 30 min, and the normal rate of DNA synthesis was not reestablished until about 60 min after irradiation. When a sample of these same cells was switched to RM immediately after UV-irradiation, there was only a slight slowing of DNA synthesis, and the normal rate of synthesis was reestablished by 60 min. An additional mmrA mutation or growth retardation by valine blocked both this extra DNA synthesis in RM, and the inhibitory effect of RM on survival. These findings suggest that the absence of a marked delay in DNA synthesis observed in RM may be responsible for the inhibitory effect of RM on the survival of UV-irradiated excision-deficient cells. Two hypotheses, which are not mutually exclusive, are propose and supported by data to explain why a fast rate of DNA synthesis after UV-irradiation partially inhibits postreplication repair and enhances cell lethality.

Induction of umuC+ gene expression in Escherichia coli irradiated by near ultraviolet light.

Abstract— The induction of umu+ gene expression caused by irradiation with near ultraviolet light (BLB; black light blue) was studied in Escherichia coli K‐12 strains with special reference to the effects of SOS repair deficiencies. The umuC+ gene expression was measured as the enzymic activity of (J‐galactosidase which is regulated by the promoter of the umuC+ operon carried in a plasmid DNA carrying a promoter of umuC* operon, a umuD+ gene and a umuC+‐lacZ+ gene fusion. A high induction of the umuC+ gene expression was observed in the uvrA cells in the case of BLB or UV irradiation as compared with the parental wild‐type cells. Caffeine inhibited the induction of the umuC* gene expression due to BLB or UV irradiation in both strains. There was very little induction in lexA and recA mutants. In contrast with UV irradiation, there was no killing of cells by BLB irradiation in any strain (wild, uvrA, lexA and recA). Possible implications of the present experimental results were discussed.

Photoreactivation of UV damage in Escherichia coli uvrA6: Lethality is more effectively reversed than either premutagenic lesions or SOS induction

The effect of cyclobutyl pyrimidine dimers on cytotoxicity, induction of synthesis of the RecA and UmuC proteins, and mutagenesis was studied in Escherichia coli uvrA6 cells possessing excess amounts of photoreactivating enzyme. Exposure of 254 nm ultraviolet-irradiated (10 J/m 2) cells to radiation from daylight fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence-dependent manner, up to about 90% reduction at 5 min exposure. Of the lethal ultraviolet damage, 85% was photoreactivable (i.e. cyclobutyl pyrimidine dimers) and 15% was non-photoreactivable. An incident fluence of 1 J/m 2 resulted in approximately a 5-fold increase in the synthesis of the RecA and UmuC proteins, as compared to the spontaneous level. If the UV-irradiated cell suspensions were illuminated with a fluorescent lamp at a dose which resulted in the full photoreactivation of viability, the yields of both proteins were reduced to 60% of the non-photoeractivated control cells. Furthermore, photoreactivation was shown to be more effective in the repair of lethal damage than in the repair of premutational damage. These experiments suggest that, among lethal damages, non-photoreactivable damage plays a more important role in both induction of the SOS functions and mutagenesis in uvrA6 cells than do cyclobutyl pyrimidine dimers.