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.     

Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli.

A system of cells made permeable by treatment with high concentrations of surcrose (plasmolysis) has been exploited to study the excision repair of ultraviolet-irradiated deoxyribonucleic acid in Escherichia coli. It is demonstrated that adenosine 5'-triphosphate is required for incision breaks to be made in the bacterial chromosome as well as in covalently closed bacteriophage lambda deoxyribonucleic acid. After plasmolysis, uvrC mutant strains appear as defective in the incision step as the uvrA-mutated strains. This is in contrast to the situation in intact cells where uvrC mutants accumulate single-strand breaks during postirradiation incubation. These observations have led to the proposal of a model for excision repair, in which the ultraviolet-specific endonuclease, coded for by the uvrA and uvrB genes, exists in a complex with the uvrC gene product. The complex is responsible for the incision and possibly also the excision steps of repair. The dark-repair inhibitors acriflavine and caffeine are both shown to interfere with the action of the adenosine 5'-triphosphate-dependent enzyme.     

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.     

Impaired incision of ultraviolet-irradiated deoxyribonucleic acid in uvrC mutants of Escherichia coli.

The production of single-strand breaks in the deoxyribonucleic acid of irradiated uvrC mutants of Escherichia coli K-12 was studied both in vivo and in vitro. In vivo, uvrC mutants displayed a slow accumulation of breaks after irradiation, and in this respect appeared different from uvrA mutants, in which very few breaks could be detected. The breakage observed in uvrC mutants differed from that observed in wild-type strains in both the slow rate of break accumulation and the very limited dose response. The behavior of the uvrC lig-7(Ts) double mutant was shown not to be consistent with the suggestion of ligase reversal as the explanation for the lower rate and limited dose response of break formation observed in ultraviolet-irradiated uvrC mutants in vivo. Rather, there appeared to be a real defect in incision. In toluene-treated cells, we studied the effect of the ligase inhibitor nicotinamide mononucleotide on strand incision. Whereas uvrC mutants displayed more strand breakage in the presence of this inhibitor, the same amount of breakage was seen in uvrA mutants, and as such the breakage could be judged as not due to the main excision repair pathway. Experiments using a cell-free system comprising the partially purified uvr+ gene products demonstrated clearly that there is a requirement for the uvrC+ gene product for strand incision. We suggest that in vivo in the absence of the uvrC+ gene product, a partial analog of this protein may allow some abnormal incision.     

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.     

Selectivity of the excision of alkylation products in a xeroderma pigmentosum-derived lymphoblastoid line.

Lymphoblastoid cell derived from a complementation group C xeroderma patient were unable to remove 06-methyl guanine residues formed in DNA by treatment of cells with low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The xeroderma cells were competent in their ability to excise 3-methyl adenine adducts. MNNG treatment induced excision repair in the xeroderma line and in addition the treatment resulted in the presence of numerous single-strand breaks in the DNA. The single gene, UV-excision-defective mutants of Escherichia coli, uvrA and uvrB, are able to excise MNNG-induced 06-methyl guanine adducts indicating that excision of this compound is not due to operation of UV endonuclease system.     

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.     

New insights on how nucleotide excision repair could remove DNA adducts induced by chemotherapeutic agents and psoralens plus UV-A (PUVA) in Escherichia coli cells

Chemotherapeutic agents such as mitomycin C or nitrogen mustards induce DNA inter-strand cross-links (ICL) and are highly toxic, thus constituting an useful tool to treat some human degenerative diseases, such as cancer. Additionally, psoralens plus UV-A (PUVA), which also induce ICL, find use in treatment of patients afflicted with psoriasis and vitiligo. The repair of DNA ICL generated by different molecules involves a number of multi-step DNA repair pathways. In bacteria, as in eukaryotic cells, if DNA ICL are not tolerated or repaired via nucleotide excision repair (NER), homologous recombination or translesion synthesis pathways, these DNA lesions may lead to mutations and cell death. Herein, we bring new insights to the role of Escherichia coli nucleotide excision repair genes uvrA, uvrB and uvrC in the repair of DNA damage induced by some chemotherapeutic agents and psoralen derivatives plus UV-A. These new observations point to a novel role for the UvrB protein, independent of its previously described role in the Uvr(A)BC complex, which could be specific for repair of monoadducts, intra-strand biadducts and/or ICL.     

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.     

A multicopy phr-plasmid increases the ultraviolet resistance of a recA strain of Escherichia coli

It has been previously reported that the ultraviolet sensitivity of recA strains of Escherichia coli in the dark is suppressed by a plasmid pKY1 which carries the phr gene, suggesting that this is due to a novel effect of photoreactivating enzyme (PRE) of E. coli in the dark (Yamamoto et al., 1983a). In this work, we observed that an increase of UV-resistance by pKY1 in the dark is not apparent in strains with a mutation in either uvrA, uvrB, uvrC, lexA, recBC or recF. The sensitivity of recA lexA and recA recBC multiple mutants to UV is suppressed by the plasmid but that of recA uvrA, recA uvrB and recA uvrC is not. Host-cell reactivation of UV-irradiated lambda phage is slightly more efficient in the recA/pKY1 strain compared with the parental recA strain. On the other hand, the recA and recA/pKY1 strains do not differ significantly in the following properties: Hfr recombination, induction of lambda by UV, and mutagenesis. We suggest that dark repair of PRE is correlated with its capacity of excision repair.     

Repair of Radiation-Induced Damage in Escherichia coli II. Effect of rec and uvr Mutations on Radiosensitivity, and Repair of X-Ray-Induced Single-Strand Breaks in Deoxyribonucleic Acid

Strains of Escherichia coli K-12 mutant in the genes controlling excision repair (uvr) and genetic recombination (rec) have been studied with reference to their radiosensitivity and their ability to repair X-ray-induced single-strand breaks in deoxyribonucleic acid (DNA). Mutations in the rec genes appreciably increase the radiosensitivity of E. coli K-12, whereas uvr mutations produce little if any increase in radiosensitivity. For a given dose of X-rays, the yield of single-strand breaks has been shown by alkaline sucrose gradient studies to be largely independent of the presence of rec or uvr mutations. The rec(+) cells (including those carrying the uvrB5 mutation) could efficiently rejoin X-ray-induced single-strand breaks in DNA, whereas recA56 mutants could not repair these breaks to any great extent. The recB21 and recC22 mutants showed some indication of repair capacity. From these studies, it is concluded that a correlation exists between the inability to repair single-strand breaks and the radiosensitivity of the rec mutants of E. coli K-12. This suggests that unrepaired single-strand breaks may be lethal lesions in E. coli.     

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.     

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.     

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.     

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

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.     

Influence of chromosome integrity on Escherichia coli cell division.

The antitumor agent cis-platinum(II)diamminodichloride (PDD) caused wild-type and recA+ deoxyribonucleic acid (DNA) repair-deficient mutant cells of Escherichia coli K-12 to grow as long, multinucleated filaments. At 5 micrograms/ml, the times required for reduction of viability to 37% for wild-type, polA, recB,C, uvrA, and recA organisms were > 200, 200, 120, 25, and 5 min, respectively. Only recA cells exhibited @reckless" degradation of DNA at this concentration of PDD. As shown by sedimentation in alkaline sucrose gradients, generation of single-strand breaks in DNA of the remaining organisms was a major consequence of growth in PDD. Upon incubation in fresh medium after removal of the compound and storage for 4 h at 4 degrees C, a respective lag of 3, 4, 6, and 9 h occurred before filaments of wild-type, polA, recB,C, and uvrA cells commenced cell division. Maintenance at 4 degrees C, which evidently delayed postshift initiation of chromosome replication, was only essential for fragmentation of uvrA filaments. In all cases, these periods of division delay corresponded to those required for restoration of normal chromosomal molecular weight as determined in alkaline sucrose gradients.     

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.     

Inhibition of deoxyribonucleic acid repair in Escherichia coli by caffeine and acriflavine after ultraviolet irradiation.

The effects of caffeine and acriflavine on cell survival, single-strand deoxyribonucleic acid break formation, and postreplication repair in Escherichia coli wild-type WP2 and WP2 uvrA strains after ultraviolet irradiation was studied. Caffeine (0.5 mg/ml) added before and immediately after ultraviolet irradiation inhibited single-strand deoxyribonucleic acid breakage in wild-type WP2 cells. Single-strand breaks, once formed, were no longer subject to repair inhibition by caffeine. At 0.5 to 2 mg/ml, caffeine did not affect postreplication repair in uvrA strains. These data are consistent with the survival data of both irradiated WP2 and uvrA strains in the presence and absence of caffeine. In unirradiated WP2 and uvrA strains, however, a high caffeine concentration (greater than 2 mg/ml) resulted in gradual reduction of colony-forming units. At a concentration insufficient to alter survival of unirradiated cells, acriflavine (2 microgram/ml) inhibited both single-strand deoxyribonucleic acid breakage and postreplication repair after ultraviolet irradiation. These data suggest that although the modes of action for both caffeine and acriflavine may be similar in the inhibition of single-strand deoxyribonucleic acid break formation, they differ in their mechanisms of action on postreplication repair.