Overexpression of enzymes that repair endogenous damage to DNA.

A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin. Efficient repair mechanisms have evolved to cope with this. The main repair pathway involved in repair of endogenous damage is DNA base excision repair. In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine. In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities. In some cases (e.g. O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g. alkyl N-purine glycosylase or DNA polymerase beta) were detrimental and even produced a genome instability phenotype. The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.     

DNA damage, prophage induction and mutation by furazolidone

Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that furazolidone treatment of Vibrio cholerae cells produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the reaction obeyed a first order relation. Sensitivities of the Escherichia coli strains to the lethal action of the drug were in the order: AB 2480(uvr- rec-) greater than AB 2463(rec-) greater than AB 1886(uvr-) greater than AB 1157(repair proficient) or AB 4401(wild type). Furazolidone was 'Rec test' positive, produced dose-dependent prophage induction in E. coli cells and also dose-dependent streptomycin-resistance forward mutation in V. cholerae cells. The quantitative aspect and also the mode of furazolidone action on DNA were discussed.     

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

Summary 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 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 contranst 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. Using the BuG data reported here and in our previous paper, we calculate that BuG pairs with a thymine residue 0.5%–0.62% of the time during replication in vivo, and that BuG markedly inhibits replication of the strand that contains it. Because of the complication introduced by alkyl transfer repair, similar calculations for MeG cannot be made from the current data.Abbreviations MeG and BuG O⁶-methyl-or O⁶-n-butyl-guanine moiety in X DNA (in each case, the plus strand nucleotide is specified first) - form I'DNA relaxed, covalently closed, circular, double-stranded DNA - Wt wild-type phenotype - Am amber phenotype - pfu plaque forming units - MNNG N-methyl-N'-nitro-N-nitrosoguanidine X mutants are named by designating the gene, the type of mutation (e.g. ms=missense), the codon number, the mutant codon and the new amino acid (where pertinent) in that order (e.g. XGam3) carries an amber in the third codon of gene G, and should not be confused with the classical am3 mutant used in the older literature to designate what is now known to be XEam7     

Site-specific mutagenesis induced by single O6-alkylguanines (O6-n-propyl, O6-n-butyl, O6-n-octyl) in vivo.

The mutagenic activity of a series of longer chain O6-n-alkylguanine residues (O6-n-propyl, O6-n-butyl, O6-n-octyl) has been analyzed using a plasmid molecule (pUC 9) in which single O6-alkylguanines were positioned in the unique Pstl recognition site by shot gun ligation (Nucleic Acids Res. 13, 3305-3316 (1985)) of overlapping synthetic oligonucleotides. After transfection of these vectors into E. coli cells having normal DNA repair systems, progeny plasmids were produced, of which 2.6%, 2.8% and 4.3% were mutated in their Pstl site when containing O6-n-propylguanine, O6-n-butylguanine, O6-n-octylguanine, respectively. DNA sequence analysis of mutant plasmid genomes revealed that O6-n-propylguanine and O6-n-butylguanine induced exclusively G-->A transitions located specifically at the preselected site. O6-n-octylguanine induced apart from G-->A transitions (70%) also targeted G-->T transversions (30%). These results indicate that the mutation frequency of longer chain O6-alkylguanines can be substantial in cells with normal repair systems and that the mutation pattern depends on the nature of the alkyl group.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

DNA damage by 5-nitro-2-furylacrylic acid, a nitrofuran derivative

5-Nitro-2-furylacrylic acid (5-NFA) caused dose dependent inhibition of growth of Escherichia coli K-12 strain AB 2480 (uvr-, rec-), the 37% (D37) and 10% (D10) survival doses being 1.0 microgram/ml.h and 1.75 micrograms/ml.h, respectively. Although much higher doses of drug were required to achieve comparable inhibition of growth of E. coli strain 1157 (repair proficient), significant filamentation of these cells was produced by treatment with 1.0 microgram/ml 5-NFA for 4 hr. Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that 5-NFA treatment of E. coli strain AB 2480 produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the initial part of the reaction obeyed a first order relation. 5-NFA also produced dose-dependent increase of prophage induction in E. coli strain GY 5027: envA, uvrB, ampA1, strA (lambda). The implications of the action of 5-NFA on DNA in relation to the induction of 'SOS' functions and carcinogenesis were discussed.     

Expression of the ogt gene in wild-type and ada mutants of E. coli.

O6-alkylguanine (O6-AlkG) DNA alkyltransferase (ATase) and alkylphosphotriester (AlkP) ATase activity have been quantitated individually in extracts of various E. coli strains by means of ATase specific DNA substrates. O6-AlkG ATase activity was higher than AlkP ATase activity in the wild-type strains F26, AB1157 and SB229 and in the ada- mutants PJ1, PJ3, PJ5 and PJ6 indicating a 5-70 times higher level of expression of the ogt gene than the ada gene. The ada- mutant strains BS23, BS73 and GW5352 expressed O6-AlkG ATase but not AlkP ATase activity indicating expression only of the ogt gene. Southern analysis of DNA from F26, BS23, BS73, PJ1 and GW5352 showed a consistent pattern of hybridisation to an ogt probe but not to an ada probe. Exposure of E. coli to adaptive doses of N-methyl-N-nitro-N-nitroso-guanidine (MeNNG) caused an increase in AlkP ATase activity in F26, AB1156, SB229, PJ1, PJ3, PJ5 and PJ6. O6-AlkG ATase activity also increased in F26, AB1157 and SB229 but decreased to almost undetectable levels in all other strains examined except PJ3 where it remained constant. MeNNG increased ada mRNA abundance in F26 but no ada mRNA was detected in BS23, BS73 or GW5352: there was no evidence for increased ogt mRNA in any of the strains examined. In a limited survey, other bacterial strains have been shown to possess an ogt-like ATase activity.     

UV-induced alleviation of K-specific restriction of bacteriophage lambda.

A partial release of K-specific restriction of phage lambda grown in Escherichia coli C was observed when E. coli K strains AB1157 (having wild-type repair of UV-produced DNA damage) and AB1886 (uvrA) were irradiated with UV light before infection. The effect occurred in AB1886 at lower UV fluences than it did in AB1157. Little or no release of restriction was observed when AB2463 (recA) or AB2494 (lex-1) was used. Such release of restriction appears to be another of the UV-induced phenomena associated with "SOS" repair.     

Function of domains of human O6-alkylguanine-DNA alkyltransferase

O6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects from alkylating agents by converting O6-alkylguanine to guanine forming S-methylcysteine in the AGT protein. The crystal structure of human AGT shows clearly the presence of two domains. The N-terminal domain contains a bound zinc atom, and zinc binding confers a mechanistic enhancement to repair activity, but this domain has no known function. The C-terminal domain contains all residues so far implicated in alkyl transfer including the cysteine acceptor site (Cys145), the O6-alkylguanine binding pocket, and a DNA binding domain. We have expressed and purified the two domains of human AGT separately. The C-terminal domain was totally inactive in vitro, but good activity forming S-alkylcysteine at Cys145 was obtained after recombination with the N-terminal domain via a freeze-thawing procedure. This suggests that the N-terminal domain plays a critical structural role in maintaining an active configuration of the C-terminal domain. However, this C-terminal domain alone had activity in protecting against the cytotoxic and mutagenic activity of the methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) when expressed in Escherichia coli cells lacking endogenous AGT, suggesting that other proteins can fulfill this function. Remarkably, the free N-terminal domain of hAGT was able to repair O6-alkylguanine in vitro via alkyl transfer provided that zinc ions were present. The N-terminal domain was also able to produce moderate protection from MNNG when expressed in E. coli. This cryptic Zn2+-dependent DNA repair activity may be relevant to the evolution and function of AGTs.     

Survival and the repair of single-stranded DNA breaks in gamma-irradiated Escherichia coli cells adapted to methylmethane sulfonate]

The survival and repair of single-strand breaks of DNA in gamma-ray-irradiated E. coli adapted to MMS (20 mkg/ml during 3 hours) have been investigated. It is shown that the survival of adapted bacteria of radioresistant strains B/r, H/r30, AB1157 and W3110 pol+ increases with DMF (dose modification factor) ranging within 1.4-1.8 and in radiosensitive strains Bs-1, AB1157 recA13 and AB1157 lexA3 with DMF ranging within 1.3-1.4, and does not change in strains with mutation in polA gene P3478 polA1 and 016 res-3. There is no increase in radioresistance during the adaptation to MMS under the action of the protein synthesis inhibitor chloramphenicol. The increase in radioresistance during the adaptation to MMS correlates with the acceleration of repair of gamma-ray-induced single-strand breaks in the radioresistant strains B/r and W3110 pol+ and with the appearance of the ability to repair some part of DNA single-strand breaks in the mutant Bs-1, which beyond the adaptation to MMS does not repair these damages. The incomplete reparability of DNA single-strand breaks in P3478 polA1 strain cells, both adapted and non-adapted to MMS, is equal.     

Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.     

Survival and repair of single-stranded DNA breaks after gamma-irradiation of Escherichia coli cells depending on the presence of plasmids pKN101 and COIIB-P9

Plasmids, pKM101 and ColIb-P9, present in an autonomous state in E. coli AB 1157, JC5519, and P3478 cells at the stationary and logarithmic phases of growth, somewhat sensitize the cells to the lethal effect of gamma-radiation and do not influence the radiosensitivity of B/r, Bs-1 gamma R, Bs-1, and W3110 cells. The efficiency of repair of gamma-ray-induced DNA single-strand breaks in AB1157 and P3478 cells containing plasmids is somewhat lower than that in the same non-plasmid strains.     

Biological effects of rutin on the survival of Escherichia coli AB1157 and on the electrophoretic mobility of plasmid PUC 9.1 DNA.

The use of natural products as medicines is growing in the world. The rutin, a compound isolated from Ruta graveolens, is a flavonoid, which has been suggested to have antioxidant properties and to reduce the triacylglycerol levels. In this study, plasmid desoxyribonucleic acid (DNA) was exposed to rutin (0.33, 10, 20, 30 microg/ml) in presence of stannous chloride (SnCl2), a reducing agent widely used to obtain radiopharmaceuticals labeled with technetium-99m. Samples of the plasmid DNA were analyzed through agarose gel electrophoresis. E. coli AB1157 culture was also incubated with rutin (3, 30, 50, 100 microg/ml) and the survival fractions were calculated. The results show that the rutin, in these concentrations, is not capable of: i/ damaging the DNA, ii/ protecting the DNA from the SnCl2 redox action, and iii/ inactivating the E. coli AB1157 culture. The analysis of our data indicates that rutin do not present toxic activity in the evaluated systems.     

A common pathway for protection of bacteria against damage by solar UVA (334 nm, 365 nm) and an oxidising agent (H2O2)

Pre-exposure of growing bacterial populations to low concentrations of hydrogen peroxide (H2O2) protects a repair-proficient strain of Escherichia coli (AB1157) very strongly and a rec A strain (AB2463) to a lesser extent from the lethal action of subsequent exposure to 5 mM H2O2 in buffer. The conditioning procedure also protects AB1157 and AB2463 from the toxic effects of UVA (334 nm, 365 nm) radiation but not UVB (313 nm) or UVC (254 nm) radiations. Pretreatment of growing AB1157 with low fluences of UVA (365 nm) radiation leads to the induction of resistance to H2O2, an effect which apparently requires protein synthesis. As in a previous report, the treatment of growing populations with low concentrations of H2O2 enhanced the resistance of such populations to H2O2 challenge in the growth medium. However, when H2O2 (+ Cu2+)-treated bacteriophage were subsequently infected into AB1157 under optimal inducing conditions, their resistance was not enhanced relative to infection into untreated bacteria. We conclude that the primary mechanism for the inducible effects observed could be the induction of H2O2 scavenging activity by low concentrations of H2O2 either introduced into the growth medium directly or produced by low fluences of UVA irradiation.     

Radiation induced-like effects of four home bleaching agents used for tooth whitening: effects on bacterial cultures with different capabilities of repairing deoxyribonucleic acid (DNA) damage.

"Home bleaching" methods are commonly used in dentistry to correct tooth discoloration. This technique employs carbamide peroxide, in several concentrations, where the active component is hydrogen peroxide. In patients undertaking this treatment, this exposure can cause biological effects mainly due to the activity of hydrogen peroxide. Hydrogen peroxide is associated with effects induced by chemical (natural and synthetic substances) and physical agents (ionizing radiations). We have evaluated the cytotoxic effects of four commercial dental bleaching agents: Insta-Brite, Karisma, Opalescence and Whiteness. We have studied the effects of these agents on the survival of different E. coli strains with various capabilities to repair damages on the deoxyribonucleic acid (DNA): AB1157 (wild type), AB2463 (recA) and BW9091 (xthA). To determine the effect of the bleaching agents on the survival of E. coli AB1157, AB2463 and BW9091, cultures in exponential growth-phase were incubated with the bleaching agent or with 0.9% NaCl, as a control. After plating, the survival fractions were determined. The bleaching agents tested decreased the survival fractions of all strains studied and the E. coli BW9091 was the most sensitive and, moreover, these bleaching agents are capable of inducing damage to the DNA molecule. In conclusion, our results indicate that dental bleaching agents can generate biological effects like the ionizing radiations, and we suggest that dental professionals involved in bleaching to correct tooth discoloration, should control the clinical environment strictly, thus preventing contact between the oral mucosa/gingival tissues and the bleaching agents.     

Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid.

A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.     

Enhanced survival of gamma-irradiated Escherichia coli following pretreatment with dithiothreitol

Survival of three strains of Escherichia coli K12 was studied with respect to radiation protection by dithiothreitol (DTT). The three strains compared were AB2462 recA, AB2470 rec21 and their DNA repair-competent prototype, AB1157. The strains were incubated in 10 mmol dm-3 DTT for 60 min and allowed an expression period for SOS functions to appear which may have been induced by DTT. Following the expression period the DTT-incubated cells and incubated control cells were irradiated. When AB1157 cells were pretreated with chloramphenicol (200 micrograms cm-3) for a period of 30 min prior to addition of the induction media no increase in survival was seen. When catalase (0.1 mg cm-3) was added to the AB1157 cells prior to the induction media a decrease in the degree of induction was noted with an enhancement ratio (ER) of 0.893 (ER-1 = 1.12). Furthermore, DTT-treated AB2462 and AB2470 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with or without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a dose modification factor (DMF) of 1.7 with DTT present and 1.3 with pretreatment; (2) the rec mutants showed no change in survival at any dose with a DMF of approximately 1.0. Results indicate that, using our protocol, inducible repair is of more importance than free radical scavenging by DTT. Furthermore, DTT-treated AB2462 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with and without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a DMF of 1.7 with DTT present during irradiation and 1.3 with only pretreatment; (2) the recA and recB mutants showed no change in cell survival at any dose with a DMF of approximately 1.0. Results indicate that, using our pretreatment protocol, inducible repair is of more importance in protection than free radical scavenging by DTT.     

Release of normal bases from intact DNA by a native DNA repair enzyme.

Base excision repair is initiated by DNA glycosylases removing inappropriate bases from DNA. One group of these enzymes, comprising 3-methyladenine DNA glycosylase II (AlkA) from Escherichia coli and related enzymes from other organisms, has been found to have an unusual broad specificity towards quite different base structures. We tested whether such enzymes might also be capable of removing normal base residues from DNA. The native enzymes from E.coli, Saccharomyces cerevisiae and human cells promoted release of intact guanines with significant frequencies, and further analysis of AlkA showed that all the normal bases can be removed. Transformation of E. coli with plasmids expressing different levels of AlkA produced an increased spontaneous mutation frequency correlated with the expression levels, indicating that excision of normal bases occurs at biologically significant rates. We propose that the broad specificity 3-methyladenine DNA glycosylases represent a general type of repair enzyme 'pulling' bases in DNA largely at random, without much preference for a specific structure. The specificity for release of damaged bases occurs because base structure alterations cause instability of the base-sugar bonds. Damaged bases are therefore released more readily than normal bases once the bond activation energy is reduced further by the enzyme. Qualitatively, the model correlates quite well with the relative rate of excision observed for most, if not all, of the substrates described for AlkA and analogues.