The repair of psoralen monoadducts by the Escherichia coli UvrABC endonuclease.

We have examined the interactions of UvrABC endonuclease with DNA containing the monoadducts of 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP). The UvrA and UvrB proteins were found to form a stable complex on DNA that contains the psoralen monoadducts. Subsequent binding of UvrC protein to this complex activates the UvrABC endonuclease activity. As in the case of incision at pyrimidine dimers, a stable protein-DNA complex was observed after the incision events. For both 8-MOP and TMP, the UvrABC endonuclease incised the monoadduct-containing strand of DNA on the two sides of the monoadduct with 12 bases included between the two cuts. One incision was at the 8th phosphodiester bond on the 5' side of the modified base. The other incision was at the 5th phosphodiester bond 3' to the modified base. The UvrABC endonuclease incision data revealed that the reactivity of psoralens is 5'TpA greater than 5'ApT greater than 5'TpG.     

SOS-independent mutagenesis in lacZ induced by methylene blue plus visible light

Summary In vitro photosensitization by visible light in the presence of methylene blue (MB-light) produces lesions in M13mpl8 lacZ phage DNA, the lethal and mutagenic potential of which was analyzed after transfection into various bacterial hosts. Mutagenesis was determined with a forward mutation assay using the lacZ gene of M13mp18 as a target. When, MB-light-treated double-stranded (ds) M13mp18 DNA was used to transfect wild-type cells which were not induced for SOS functions, a fivefold increase in mutation frequency was observed at 10% survival compared to that observed with untreated DNA. Mutation frequency obtained with MB-light-treated ds M13mp18 DNA was greater when transfected into the uvrA fpg-1 double mutant than that seen in uvrA, fpg-1, or umuC single mutants or in the wild-type. Sequence analysis shows that in the wild-type strain, MB-light treatment of ds M13mp18 DNA results mostly in single base substitutions. The most frequent base change is the GCTA transversion. MB-light treatment of single-stranded (ss) M13mp18 DNA also results in an increased mutation frequency after transfection into the wild-type strain, yielding mostly GT transversions. Our results show that MB-light-induced mutagenesis is at least partially independent of the induction of SOS functions in Escherichia coli. The mutation spectra suggest that 8-oxo-7,8-dihydroguanine is the major promutagenic lesion in DNA.     

Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein.

The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated. DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA. Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. Kinetic studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde). Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyuracil, were not excised. Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature. The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage. Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate. These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.     

Repair of damage induced by ultraviolet light in DNA polymerase-defective Escherichia coli cells

Cells of the Escherichia coli mutant polA1, which lack DNA polymerase activity in vitro, are four times as sensitive as wild-type to ultraviolet irradiation. Cells of the mutant uvrA6, which are unable to excise dimers, are 12 times as sensitive as wild-type. We have shown that the double mutant polA1 uvrA6 is only slightly more sensitive to u.v. than the uvrA6 single mutant and conclude, therefore, that the u.v. sensitivity associated with the defect in DNA polymerase is primarily the result of a reduction in the efficiency of the excision-repair pathway. Observations on the effect of u.v. irradiation on the ability of polA1 cells to support the growth of phage λ suggest that the post-u.v. repair function of polymerase is subsequent to the action of the uvr⁺ gene products. Evidence is presented that the recA repair system is involved in excision-repair in polA1 cells, and we propose that it can substitute for DNA polymerase in repairing the gaps produced by dimer excision. This would account for the relatively slight effect of the polA1 mutation on u.v. sensitivity.     

Cell surface alterations induced by methylene blue and direct electric current in Escherichia coli.

Cell surface properties, including hydrophobicity, zeta potential, carbohydrate and fatty acid components, were altered on treatment of E. coli K12 with methylene blue (MB) and direct electric current (DC). The treatment of fimbriated E. coli cells with MB greatly increased the agglutination of yeast cells when compared to untreated bacteria. However, this increased agglutination was markedly reduced when the bacteria were treated with MB plus DC. These results suggest that MB modifies cell surface components in the absence of light and these alterations are more pronounced when cells are treated simultaneously with MB and DC.     

ATPase activity of the UvrA and UvrAB protein complexes of the Escherichia coli UvrABC endonuclease.

We have analyzed the ATPase activity exhibited by the UvrABC DNA repair complex. The UvrA protein is an ATPase whose lack of DNA dependence may be related to the ATP induced monomer-dimer transitions. ATP induced dimerization may be responsible for the enhanced DNA binding activity observed in the presence of ATP. Although the UvrA ATPase is not stimulated by dsDNA, such DNA can modulate the UvrA ATPase activity by decreases in Km and Vm and alterations in the Ki for ADP and ATP-gamma-S. The induction of such changes upon binding to DNA may be necessary for cooperative interactions of UvrA with UvrB that result in a DNA stimulated ATPase for the UvrAB protein complex. The UvrAB ATPase displays unique kinetic profiles that are dependent on the structure of the DNA effector. These kinetic changes correlate with changes in footprinting patterns, the stabilization of protein complexes on DNA damage and with the expression of helicase activity.     

DNA damage by 8-methoxypsoralen plus near ultraviolet light (PUVA) and its repair in Escherichia coli: genetic analysis

Mutants of Escherichia coli, hyper-resistant and sensitive to 8-methoxypsoralen plus near ultraviolet light (PUVA) have been isolated and studied. Results show that a mutation, located at 57.2 min on the linkage map of E. coli, is responsible for the hyper-resistant phenotype. It is also responsible for the synthesis of a 55-kdal protein in high concentrations. In a wild-type cell the synthesis of this enzyme is inducible by mitomycin C. There are indications that the mutation may have occurred in a regulatory gene, puvR, and as a result the operon, including a putative puvA gene (the structural gene for the synthesis of the 55-kdal protein), is expressed constitutively. A model for the control of the PUV operon is proposed.     

Mutagenic DNA repair in Escherichia coli. XVI. Mutagenesis by ultraviolet light plus delayed photoreversal in recA strains

Mutagenesis was demonstrable after delayed photoreversal of UV-irradiated strains carrying a recA deletion indicating that RecA protein is not essential for the misincorporation process that is revealed by delayed photoreversal. Moreover, the data suggest that RecA protein actually depresses misincorporation to varying extents depending on the recA allele. No delayed photoreversal was demonstrable in reA1 or recA56 bacteria unless the lexA102(ind-) allele was also present. It is suggested that the level of these RecA proteins may be lower in the lexA102(ind-) strains thus minimising their depressive effect. Delayed photoreversal mutagenesis in strains carrying the recA441 allele was not affected by either adenine or guanosine plus cytidine, substances which affect the proteolytic activity of RecA441 protein.     

Inducing local DNA damage by visible light to study chromatin repair

Dynamics of DNA repair and recruitment of repair factors to damaged DNA can be studied by live cell microscopy. DNA damage is usually inflicted by a laser beam illuminating a DNA-interacting photosensitizer in a small area of the nucleus. We demonstrate that a focused beam of visible low intensity light alone can inflict local DNA damage and permit studies of DNA repair, thus avoiding potential artifacts caused by exogenous photosensitizers.     

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction.     

Escherichia coli Fpg glycosylase is nonrendundant and required for the rapid global repair of oxidized purine and pyrimidine damage in vivo

Endonuclease (Endo) III and formamidopyrimidine-N-glycosylase (Fpg) are two of the predominant DNA glycosylases in Escherichia coli that remove oxidative base damage. In cell extracts and purified form, Endo III is generally more active toward oxidized pyrimidines, while Fpg is more active towards oxidized purines. However, the substrate specificities of these enzymes partially overlap in vitro. Less is known about the relative contribution of these enzymes in restoring the genomic template following oxidative damage. In this study, we examined how efficiently Endo III and Fpg repair their oxidative substrates in vivo following treatment with hydrogen peroxide. We found that Fpg was nonredundant and required to rapidly remove its substrate lesions on the chromosome. In addition, Fpg also repaired a significant portion of the lesions recognized by Endo III, suggesting that it plays a prominent role in the global repair of both purine damage and pyrimidine damage in vivo. By comparison, Endo III did not affect the repair rate of Fpg substrates and was only responsible for repairing a subset of its own substrate lesions in vivo. The absence of Endo VIII or nucleotide excision repair did not significantly affect the global repair of either Fpg or Endo III substrates in vivo. Surprisingly, replication recovered after oxidative DNA damage in all mutants examined, even when lesions persisted in the DNA, suggesting the presence of an efficient mechanism to process or overcome oxidative damage encountered during replication.     

A mutant endonuclease IV of Escherichia coli loses the ability to repair lethal DNA damage induced by hydrogen peroxide but not that induced by methyl methanesulfonate.

A mutant allele of the Escherichia coli nfo gene encoding endonuclease IV, nfo-186, was cloned into plasmid pUC18. When introduced into an E. coli xthA nfo mutant, the gene product of nfo-186 complemented the hypersensitivity of the mutant to methyl methanesulfonate (MMS) but not to hydrogen peroxide (H2O2) and bleomycin. These results suggest that the mutant endonuclease IV has normal activity for repairing DNA damages induced by MMS but not those induced by H2O2 and bleomycin. A missense mutation in the cloned nfo-186 gene, in which the wild-type glycine 149 was replaced by aspartic acid, was detected by DNA sequencing. The wild-type and mutant endonuclease IV were purified to near homogeneity, and their apurinic (AP) endonuclease and 3'-phosphatase activities were determined. No difference was observed in the AP endonuclease activities of the wild-type and mutant proteins. However, 3'-phosphatase activity was dramatically reduced in the mutant protein. From these results, it is concluded that the endonuclease IV186 protein is specifically deficient in the ability to remove 3'-terminus-blocking damage, which is required for DNA repair synthesis, and it is possible that the lethal DNA damage by H2O2 is 3'-blocking damage and not AP-site damage.     

In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo.

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.     

Repair of DNA lesions induced by hydrogen peroxide in the presence of iron chelators in Escherichia coli: participation of endonuclease IV and Fpg

In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product. Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions. Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.     

Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1.

Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents. In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese. EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese. ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA. In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2. Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme. MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes. The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV. Possible mechanistic roles for the metals in these DNA repair enzymes are discussed.