Repair-proficient Strain Research Articles

Studies on the genotoxicity of endosulfan in bacterial systems

Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage λ induction assay in WP2s (λ) and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage λ in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage.

Green Fluorescent Protein (GFP) as a marker for cell viability after UV irradiation

Generation of Chinese Hamster Ovary (CHO) cell lines stably expressing green fluorescent protein (GFP) was achieved using a plasmid vector that encoded the red-shifted pCX-xGFP under the control of a strong hybrid promoter composed of a CMV enhancer and a β-actin/β-globin gene promoter. Cotransfection of the promoter-less pSV2-Neo helper plasmid transmitting neomycin resistance was followed by selection with the antibiotic G418. Constitutive GFP expression could be visualized in living and fixed cells using fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. DNA repair-proficient (AA8) and deficient (UV5) CHO strains were used for survival tests after UVC irradiation. Cells carrying the GFP construct (AA8-pGFP, UV5-pGFP) show the same response to UV irradiation (colony forming ability) as their nontransformed parental cell lines (AA8, UV5). Using GFP as a marker for cell viability, cells were harvested after certain postirradiation growth periods and the numbers of GFP expressing cells and fluorescence intensities were determined by FACS analysis. Generally, GFP fluorescence in irradiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuously (leading to a dose-dependent increase in GFP contents).

Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system

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 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 β-galactosidase. In TestD, the strains carrying different recA alleles ( recA +, rcA730 and Δ recA) 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 monoterpernoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Test 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.

Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles

Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species. Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection. We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures. The analysis involved screening for mutators among revertants of S. typhimurium histidine auxotrophs selected for the His + phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn. The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts. Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%). the his + reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds. The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype. We calculate that the frequency of mutators in the unselected population of S. typhimurium is 1–4×10 −6, an incidence of 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli.

Mutation spectra analysis suggests that N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli

To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr-ada+ogt+, and uvr-ada-ogt-). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC-->AT transitions (the mutated G being almost exclusively preceded 5' by a purine). Mutation distribution was not random. Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt(+)-specific hot-spot. Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains. Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr- strains, whereas in the uvr- background, no difference was observed between mutation spectra in ada+ogt+ versus ada-ogt- strains. Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC-->AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli.

Antimutagens reduce ofloxacin-induced bleaching in Euglena gracilis

The genotoxic effect of ofloxacin was significantly decreased by standard antimutagens (sodium selenite, ascorbic acid, butylated hydroxyanisole and butylated hydroxytoluene) in the unicellular flagellate Euglena gracilis. The antiofloxacin activity of sodium selenite was also documented by a bacterial test in which the repair-proficient strain Salmonella typhimurium TA102 was used.

Final Report on the Safety Assessment of Sodium Iodate

Sodium Iodate is an inorganic salt that is intended for use as an oxidizing agent in cosmetics, but no current uses have been reported. It is approved by the European Union for use as a preservative in rinse-off cosmetic products at concentrations no greater than 0.1%. Pure Sodium Iodate is a sufficiently strong oxidizing agent that it presents a fire risk near organic material, and it can react violently with aluminum, arsenic, carbon, copper, hydrogen peroxide, phosphorous, potassium, sulfur, and metal sulfides. Sodium Iodate is toxic to the retina; injection of 10-4 M Sodium Iodate into the vitreous of rabbit eyes inactivated the electroretinogram in 1 day. Acute toxicity studies in mice show that concentrations of 505 mg/kg delivered orally is expected to cause convulsions and death in half the animals. No mutagenic activity was seen in Ames tests. Exposure of repair proficient strains of Escherichia coli to ionizing radiation and Sodium Iodate increased the number of DNA single-strand breaks over those seen with exposure to ionizing radiation alone. Sodium Iodate combined with aflatoxin B1 showed fewer mutations in the Ames test than did aflatoxin alone. These available data were not sufficient to support the safety of Sodium Iodate for use in cosmetic formulations. Additional data were considered necessary to evaluate the safety of this ingredient, including the purpose of use and likely concentration of use in cosmetics; 28-day dermal toxicity data; and animal irritation data as a function of dose. It cannot be concluded that this ingredient is safe for use in cosmetic products until the listed safety data have been obtained and evaluated.

Mutation spectra in Salmonella of sunlight, white fluorescent light, and light from tanning salon beds: induction of tandem mutations and role of DNA repair

We evaluated the mutagenicity of sunlight (SUN), uncovered coolwhite fluorescent light (FLR), and light from a tanning salon bed (TAN) at the base-substitution allele hisG46 of Salmonella in four DNA repair backgrounds (wild type, uvrB, pKM101, and uvrB + pKM101). Approximately 80% of the radiation emitted by TAN was within the ultraviolet (UV) range, whereas only ∼ 10% of the SUN and ∼ 1% of the FLR radiation was UV. TAN emitted similar amounts of UVA and UVB, whereas SUN emitted 50–60 × and FLR emitted 5–10 × more UVA relative to UVB. Based on total dose (UV + visible), the mutagenic potency ranking was TAN > FLR > SUN. Using colony probe hybridization and PCR DNA sequence analysis, ∼ 3000 revertants were analyzed to determine the mutational specificity of the three light sources. The mutation spectra and those induced by 254-nm UV had common features. The uvrB mutation enhanced the mutagenicity of the environmental UV sources more (20–216 ×) than did the pKM101 plasmid (∼ 20 ×) relative to wild type DNA repair. All light sources induced equal proportions of transitions and transversions in excision repair-proficient strains, but they induced more transitions relative to transversions in uvrB-containing strains. The majority of the mutations were G·C → A·T transitions that were induced equally frequently at the first or second position of the CCC codon of the hisG46 allele in all strains except TA1535 ( uvrB), where SUN and FLR induced transitions preferentially at the first position, and TAN induced them preferentially at the second position. Identified or presumptive multiple mutations, which constituted the only mutational class enhanced by all three light sources in the presence of uvrB and pKM101 either alone or together, accounted for 3–5% of the induced mutations in the plasmid-containing strains, and their increases (38–82-fold) in TA100 ( uvrB, pKM101) were the highest of any mutational class. Of the TAN-induced multiple mutations, 83% ( 19 23 ) were CC → TT tandem transitions. These results show that exposures to the nonsolar environmental UV sources FLR and TAN produce mutations similar to those produced by SUN, a known carcinogen.

Expression of Chromosomal and Plasmid-mediated Resistance to Ultraviolet Light in Escherichia coli under Aerobic and Anaerobic Conditions

Previous work has shown that plasmid-mediated DNA repair, which is active against ultraviolet light-(UV-) induced damage, does not confer resistance to the DNA-damaging drug metronidazole. However, metronidazole-induced bacterial killing is only expressed under anaerobic conditions, and it may be that plasmid-encoded DNA repair does not function under these conditions. We therefore report experiments designed to determine if plasmid-mediated DNA repair is expressed anaerobically. A series of DNA repair-deficient mutants of Escherichia coli strain AB1157 was tested for sensitivity to UV after aerobic or anaerobic incubation. The repair-proficient strain and strains deficient in excision repair, recombination repair or the expression of SOS repair, were all more resistant to UV when incubated aerobically after exposure to UV. Strains deficient in the induction of SOS repair were no more sensitive whether incubated aerobically or anaerobically post-UV. Plasmid R46 mediated a high level of UV resistance in E. coli strain TK501 under anaerobic conditions, although this was lower than the level expressed when cells were incubated aerobically post-UV. The UV-sensitizing effect of plasmid R391 was expressed to a similar extent in both aerobically- and anaerobically-incubated cultures of strain TK501. These results suggest that the previously-reported lack of plasmid-mediated DNA repair activity in metronidazole-damaged TK501 cells is due to the inability of plasmid-encoded repair products to deal with metronidazole-induced damage, rather than to the inability of the repair process to function under anaerobic conditions.

Molecular analysis of CXPD mutations in the repair-deficient hamster mutants UV5 and UVL-13

The cDNA sequence of the Chinese hamster xeroderma pigmentosum group D ( CXPD) nucleotide excision repair gene was analyzed from three Chinese hamster ovary (CHO) cell lines: repair proficient strain AA8 and repair deficient, UV complementation group 2 strains UV5 and UVL-13. CXPD encodes a presumed ATP-dependent DNA helicase and is single copy in CHO lines due to the hemizygosity of chromosome 9. Comparison of the deduced wild-type AA8 CXPD protein sequence with that of the Chinese hamster V79 lung-derived cell line revealed two amino acid polymorphisms. Position 285 is glutamine in AA8 and arginine in V79, and position 298 is alanine in AA8 and threonine in V79. Comparison with the human XPD, Saccharomyces cerevisiae RAD3, and Schizosaccharomyces pombe rad15 homologs shows variability at these positons. Analysis of the CXPD sequence in the repair deficient CHO lines UV5 and UVL-13 revealed, in each case, a single base substitution resulting in an amino acid substitution. Position 116 is tyrosine in UV5 and cysteine in AA8, and the corresponding positions of XPD, RAD3, and rad15 arc cysteine. Position 615 is glutamic acid in UVL-13 and glycine in AA8, and the corresponding positions of XPD, RAD3, and rad15 are glycine. In both UV5 and UVL-13, positions 285 and 298 are glutamine and alanine, respectively, as seen in AA8. These results suggest that cysteine 116 and glycine 615 are critical to the repair function of CXPD.

Thiopyronine- and 8-methoxypsoralen-sensitized photodynamic effect on cell growth, colony forming ability and RNA synthesis in Saccharomyces mutants deficient in DNA repair.

We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8-methoxypsoralen (8-MOP) and ultraviolet A (UV-A) light, on growth, colony forming ability and RNA synthesis in a repair-proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8-MOP and UV-A repair-deficient mutants were significantly more sensitive than the repair-proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.

DNA-directed aniline mustards with high selectivity for adenine or guanine bases: mutagenesis in a variety of Salmonella typhimurium strains differing in DNA-repair capability

Two closely-related aniline monomustards ( 1 and 2), linked to a DNA-targeting acridine chromophore by a linker chain of different length, show high selectivity for alkylation of polymer DNA. The shorter-chain derivative ( 2) alkylates mainly at guanine N7 sites, while the longer-chain analogue ( 1) reacts almost exclusively at adenine N1. The biological effects of these compounds have been studied in standard Ames Salmonella typhimurium strains in order to determine the mutagenic consequences of such well-defined DNA lesions, and the effect of DNA-repair systems on them. Both compounds caused detectable mutations in strains TA1537, TA98 or TA100 and some related strains. Mutation rates were greatly enhanced in strains carrying either a uvrB deletion or the plasmid pKM101. Frameshift mutagenesis by both compounds was completely eliminated by recA deletion, in both the presence or absence of the plasmid. The adenine-selective compound ( 1) appeared more sensitive to the DNA-repair defects than the guanine-selective derivative ( 2). Additionally, only the adenine-selective compound ( 1) caused statistically significant levels of detectable mutation in the repair-proficient strains TA102, TA4001 or TA4006. The bacterial mutagenesis evidence suggests that a bulky, major groove-residing adenine lesion may be more readily recognised by DNA-repair systems, and more likely to lead to a wider range of mutagenic events, than a similar guanine lesion.

Effect of 60-Hz magnetic fields on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae

The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatic exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2–50 J/m 2). These experiments were performed using a repair-proficient strain ( RAD −), as well as a strain of yeast ( rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.

Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription.

While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia coli, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARS1 plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpb1-1, in the largest subunit of RNA polymerase II. When exponentially growing rpb1-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpb1-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpb1-1 cells was reduced to that in the nontranscribed strand. These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast.

Influence of DNA repair defects (rad1, rad52) on nitrogen mustard mutagenesis in yeast.

Nitrogen mustard (HN2) mutagenesis of a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was examined in a repair-proficient yeast strain and isogenic derivatives defective for excision (rad1) or DNA double-strand break (rad52) repair. The excision repair deficiency sensitized the cells to killing by HN2 and abolished mutation induction. Inactivation of RAD52 had no influence on the lethality of HN2 treatment but diminished the induced mutation frequency by 50% at all doses tested. DNA sequence analysis of HN2-induced SUP4-o mutations suggested that RAD52 contributed to the production of basepair substitutions at G.C sites. The rad52 defect appeared to alter the distribution of G.C-->A.T transitions in SUP4-o relative to the distribution for the wild-type strain. This difference did not seem to be due to an effect of RAD52 on the relative fractions of HN2-induced transitions at localized (flanked by A.T pairs) or contiguous (flanked by at least one G.C pair) G.C sites but instead to an influence on the strand specificity of HN2 mutagenesis. In the repair-proficient strain, the transitions showed a small bias for sites having the guanine on the transcribed strand and this preference was eliminated by inactivation of RAD52.

The distribution of UV damage in the lacI gene of Escherichia coli: Correlation with mutation spectrum

We have determined the UV (254 nm) damage distribution in the transcribed and non-transcribed strands of the i −d region of the Escherichia coli lacI gene. The locations of replication blocking lesions were revealed as termination sites of T7 DNA polymerase and/or T4 DNA polymerase 3′-5′ exonuclease. Termination products, i.e. both cyclobutane pyrimidine dimers and 6-4 photoproducts, were resolved and analysed on an automated DNA sequencer. These two major photoproducts are not randomly distributed along the gene, but occur in clusters, in longer runs of pyrimidines. We also have compared the UV damage distribution with the previously reported UV-induced base substitutions in the same region. Mutational hotspots, in both repair-deficient and repair-proficient strains of E. coli, are all located in stretches of pyrimidines, and thus correlate well the distribution of photolesions. One mutational hotspot in the wild-type strain may reflect the high frequency of closely opposed lesions. To explain the other mutational hotspots, we propose that the repair of UV lesions is impaired due to the local conformation of the DNA, which might deviate from the B-form. This hypothesis is supported by the excess of mutational hotspots in pyrimidine runs in the Uvr + strain compared to Uvr −. Runs of pyrimidines thus represent both damage- and mutation-prone sequences following UV treatment.

Induction of error-free DNA repair inEscherichia coli by Nonmutagenic Stress

Our previous studies have shown that heat shock and nutritional stress produce an increase in UV resistance and a decrease in UV-induced mutation frequency in DNA repairproficient strains ofEscherichia coli K12. The effect depends on nucleotide excision repair and requires protein synthesis. We now show that comparable changes occur after oxidation stress, exposure to ethanol, or osmotic shock, all in conditions that do not affect the natural mutation frequency. The results support the hypothesis that many unrelated, nonmutagenic treatments elicit a common protective response in these cells that involves induction of an error-free DNA excision repair system.

A sectored colony assay for monitoring mutagenesis by specific carcinogen-DNA adducts in Escherichia coli

To study the mutagenicity of various carcinogen-DNA adducts in Escherichia coli, a cassette plasmid was developed that permits positioning of specific carcinogen-modified bases within the ATG initiation codon of the lacZ' alpha-complementation gene. Adduct-induced mutations inactivate the gene and lead to formation of blue and white sectored colonies when transformants from an alpha-complementing version of E. coli strain AB1157 are grown on media containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside. In the absence of mutation, blue colonies are produced. This system has been used to measure the mutagenicity of O6-methyl-, O6-ethyl-, and O6-benzyl-2'-deoxyguanosine residues incorporated in place of the normal 2'-deoxyguanosine of the ATG initiation codon. Although a low percentage of sectored colonies was produced in this repair-proficient strain, pretreatment of the bacteria with N-methyl-N'-nitro-N-nitrosoguanidine to disable DNA repair led to a dose-dependent increase in the percentage of sectored colonies. This percentage increased as a function of modified guanine in the order O6-benzyl- less than O6-methyl- less than O6-ethyl-2'-deoxy-guanosine. The only mutations detected at the site of incorporation of these O6-substituted guanines were G-to-A transitions. This sectored colony assay system permits convenient screening of large numbers of colonies and simplifies quantification of modified-base-induced mutations whether they be single-base changes, frameshifts, insertions, or deletions.

The influence of the nucleotide excision-repair system on mutagenesis in Salmonella typhimurium LT2 after exposure to low doses of monofunctional alkylating agents

The role of nucleotide excision repair in the mutagenicity of the monofunctional alkylating agents Nmethyl- N′-nitro- N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl- N′-nitro- Nnitrosoguanidine (ENNG), and N-ethyl- N-nitrosourea (ENU) in Salmonella typhimurium was examined. The mutagenic potential of the mutagenic agents used increased in the following order: MMS < ENU < ENNG < MNNG. The results obtained confirm the involvement of nucleotide excision repair in the removal of mutagenic lesions from the DNA of S. typhimurium cells exposed to high doses of methylating as well as ethylating agents. At the low doses of ail the alkylating agents used, the nucleotide excision repairproficient strain was mutagenized more efficiently than the uvrB mutant. This phenomenon, a consequence of competition between nucleotide excision-repair enzymes and constitutive O 6-methylguanine-DNA methyltransferase, is discussed.

Immunoanalysis of ultraviolet radiation induced DNA damage and repair within specific gene segments of plasmid DNA

The region-specific heterogeneity of repairing DNA damage has been established in several biological systems. A flexible and sensitive approach, based upon DNA damage specific antibodies, is described to monitor the repair of specific lesions within discrete genomic segments. Membrane transblotted DNA restriction fragments are immunoanalyzed for the initial formation and repair of 254 nm radiation induced pyrimidine dimers. Sensitivity of dimer immunodetection increases proportional to fragment concentration and size. Antibody binding was detectable in a 0.5 kb fragment obtained from approx. 100 ng of restriction digested phage lambda DNA irradiated with 50 J m-2. Dimers within larger fragments (greater than 5 kb) could be detected at ultraviolet doses as low as 1 to 2 J m-2. To determine the occurrence of preferential repair in prokaryotic cells, damage was assessed in DNA sequences established in various Escherichia coli strains. In vivo repair of 8.9 kb vector and 6.4 and 3.2 kb gene inserts occurred with an approximate t1/2 of 45 min in UvrABC excision repair-proficient strains. Antibody binding sites were retained by DNA within repair-deficient strains. Compared to UvrABC, the repair of DNA fragments mediated by T4 endonuclease V was rapid and complete within 30 min of cellular irradiation. The efficient repair in DenV+ strain is attributable to a highly processive repair enzyme rather than to selective repair of actively replicating target genes. The results demonstrate the exceptional ability of antibodies specific for altered biomolecular lesions to map damage and repair in gene segments episomally established within cells.