Isolation of recombination-defective and UV-sensitive mutants of Bacillus megaterium.

Mutants of Bacillus megaterium QMB1551 sensitive to mitomycin C or methyl methanesulfonate were isolated and characterized phenotypically. Cell survival after UV-light and gamma-ray exposure was determined, as was transductional recombination. Of the mutants tested, three were sensitive to UV but remained recombination proficient. The UV-sensitive mutants were also reduced in host cell reactivation. At least three mutants had undetectable transduction frequencies, i.e., less than 0.3 to 1.3% of the parental strain frequencies, and so appear to be recombination deficient. Sensitivities of these mutant strains to UV light and gamma radiation were compared with those of parental B. megaterium as well as parental, recE4, recA1, uvrA19, and uvrB109 strains of Bacillus subtilis. In each case, the strains of B. megaterium, including the parental strains, showed a higher percentage of cell survival than B. subtilis.     

Dual role of NER in mutagenesis in Pseudomonas putida

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida. The genome of this organism contains two uvrA genes, uvrA and uvrA2. Genetic studies on the effects of uvrA, uvrA2, uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida. However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB, uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2, uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida-besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.     

Single-strand breakage of DNA in UV-irradiated uvrA, uvrB, and uvrC mutants of Escherichia coli.

We transduced the uvrA6, uvrB5, uvrC34, and uvrC56 markers from the original mutagenized strains into an HF4714 background. Although in the original mutagenized strains uvrA6 cells are more UV sensitive than uvrB5 and uvrC34 cells, in the new background no significant difference in UV sensitivity is observed among uvrA6, uvrB5, and uvrC34 cells. No DNA single-strand breaks are detected in UV-irradiated uvrA6 or uvrB5 cells, whereas in contrast a significant number of single-strand breaks are detected in both UV-irradiated uvrC34 and uvrC56 cells. The number of single-strand breaks in these cells reaches a plateau at 20-J/m2 irradiation. Since these single-strand breaks can be detected by both alkaline sucrose and neutral formamide-sucrose gradient sedimentation, we concluded that the single-strand breaks observed in UV-irradiated uvrC cells are due to phosphodiester bond interruptions in DNA and are not due to apurinic/apyrimidinic sites.     

Different effects of recJ and recN mutations on the postreplication repair of UV-damaged DNA in Escherichia coli K-12.

Two mutations known to affect recombination in a recB recC sbsBC strain, recJ284::Tn10 and recN262, were examined for their effects on the postreplication repair of UV-damaged DNA. The recJ mutation did not affect the UV radiation sensitivity of uvrB and uvrB recF cells, but it increased the sensitivity of uvrB recN (approximately 3-fold) and uvrB recB (approximately 8-fold) cells. On the other hand, the recN mutation did not affect the UV sensitivity of uvrB recB cells, but it increased the sensitivity of uvrB (approximately 1.5-fold) and uvrB recF (approximately 4-fold) cells. DNA repair studies indicated that the recN mutation produced a partial deficiency in the postreplication repair of DNA double-strand breaks that arise from unrepaired daughter strand gaps, while the recJ mutation produced a deficiency in the repair of daughter strand gaps in uvrB recB cells (but not in uvrB cells) and a deficiency in the repair of both daughter strand gaps and double-strand breaks in uvrA recB recC shcBC cells. Together, these results indicate that the recJ and recN genes are involved in different aspects of postreplication repair.     

Properties and regulation of the UVRABC endonuclease

This report summarizes the cloning of the uvrA, uvrB and uvrC genes of E. coli, the identification and isolation of the gene products, the regulation of the genes, and reconstitution of active UVRABC endonuclease from the individually isolated components.     

Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12.

The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9. The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells. This enhanced survival requires the host uvrA+ and uvrB+ gene products, but not the host recA+ gene product. The requirement for both homologous DNA and the uvrA+ and uvrB+ gene products suggests that a novel repair process may act on plasmid DNA. Possible mechanisms for this process are considered.     

Occurrence and loss of alkali-labile sites in newly synthesized DNA of UV-irradiated Escherichia coli K12

In pulse-labelled DNA of ultraviolet-irradiated E. coli, alkali-labile sites were detected. They do not occur in undamaged cells. These sites are produced in wild-type cells as well as in uvrA, uvrB and recA derivatives. Restoration of the synthesis of DNA molecules free of alkali-labile sites requires recA products and involves also uvrA and uvrB products. The chemical nature of alkali-labile sites and their biological function are obscure. They might be stretches of RNA that traverse the lesions, blocking DNA replication and priming recA-dependent DNA replication.     

Cross-linking studies with the uvrA and uvrB proteins of E. coli

The interactions of the uvrA and uvrB proteins with DNA have been investigated using a DNA-protein cross-linking technique. It is demonstrated that hydrolysis of ATP by the uvrA protein facilitates cross-linking of this protein to single-stranded DNA, whether the DNA is UV irradiated or not. In contrast, cross-linking to unirradiated double-stranded DNA is not facilitated by ATP hydrolysis and is in fact increased by the substitution of the non-hydrolysable analogue aTP gamma S for ATP. In the presence of ATP, a dose-dependent increase is observed in the amount of uvrA protein which can be cross-linked to UV-irradiated double-stranded DNA. Binding of uvrB protein to puvrA-DNA complexes has a stabilising effect and increases the number of complexes which can be cross-linked whether the substrate is single- or double-stranded DNA. We can find no evidence that ATP hydrolysis by uvrA protein results in unwinding of UV-damaged DNA.     

Paradoxical behaviour of pKM101; inhibition of uvr-independent crosslink repair in Escherichia coli by muc gene products

In strains of Escherichia coli deficient in excision repair (uvrA or uvrB), plasmid pKM101 muc+ but not pGW219 mucB::Tn5 enhanced resistance to angelicin monoadducts but reduced resistance to 8-methoxy-psoralen interstrand DNA crosslinks. Thermally induced recA-441 (= tif-1) bacteria showed an additional resistance to crosslinks that was blocked by pKM101. Plasmid-borne muc+ genes also conferred some additional sensitivity to gamma-radiation and it is suggested that a repair step susceptible to inhibition by muc+ gene products and possibly involving double-strand breaks may be involved after both ionizing radiation damage and psoralen crosslinks.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

Nucleotide sequence of promoter, operator and amino-terminal region of caa, the structural gene of colicin A

The nucleotide sequence of 378 bp covering the promoter-operator regions and the region coding for the N-terminal portion of the colicin A gene was determined. These assignments were made possible by the determination of the N-terminal 12 amino acids of the colicin A protein. DNA sequence homologies between operator regions of recA, lexA, uvrA, uvrB, cea and caa genes are discussed.     

Hypersensitivity of Escherichia coli Delta(uvrB-bio) mutants to 6-hydroxylaminopurine and other base analogs is due to a defect in molybdenum cofactor biosynthesis

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.     

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products.

We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo[a]pyrene diol epoxide isomers. Using the phi X174 RF DNA-E. coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells. In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells. Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts. Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate. In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions. Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding. These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts. It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Azide-induced mutagenesis in gram-negative bacteria is recA-and lexA-independent.

Azide-induced mutagenesis was investigated in Salmonella typhimurium and Escherichia coli. Azide was highly effective in inducing mutation in uvrB, uvrB recA and uvrB recB mutants of S. typhimurium. The mutagenic effect of azide was also observed in uvrA lexA mutants of E. coli K12 and E. coli B/r. These results suggest that azide-induced mutagenesis is due to mis-replication of DNA.     

Repair of helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins.

The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.     

uvrC gene function has no specific role in repair of N-2-aminofluorene adducts.

In Escherichia coli, plasmid DNA modified with N-2-aminofluorene adducts survived equally well in wild-type, uvrA, or uvrB strains. Increased sensitivity was found in uvrC and uvrD strains. Moreover, N-2-aminofluorene-mediated toxicity in the uvrC background was reversed when an additional uvrA mutation was introduced into the strain.     

Expression of Erwinia chrysanthemi ENA49 uvr gene in Escherichia coli K12 cells

The uvrA gene of Erwinia chrysanthemi ENA49 similar to uvrA gene of Escherichia coli K12 has been cloned in vivo in Escherichia coli AB1886 uvrA6 cells using the plasmid pULB113 (RP4mini Mu). The presence of pULB113 carrying uvrA gene of Erwinia in Escherichia coli K12 uvrA- cells resulted in suppression of this mutation while uvrB and uvrC are not suppressed by this locus. The genetic control of excision repair of UV-damage in Erwinia chrysanthemi ENA49 is concluded to be similar to the one in Escherichia coli K12.     

Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light.

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.