Choloroquine inhibition of repair of DNA damage induced in mammalian cells by methyl methanesulfonate

Chloroquine (ClQ) inhibited the repair of DNA damage produced in cultured rat liver cells by methyl methanesulfonate (MMS). MMS caused fragmentation of single-strand DNA in alkaline sucrose gradients. Repair of the damage was followed by observing the restoration of the normal sedimentation pattern at intervals after treatment. Repair was significant by 7 h and nearly complete at 24 h. Addition of ClQ during the repair peiod markedly reduced the rate of repair. Also, ClQ increased the lethality of MMS, which could be due to the inhibition of repair. ClQ was found to inhibit protein synthesis, but the effect on repair is probably not due entirely to this action since comparable inhibition of protein synthesis by cycloheximide produced a lesser degree of delay in repair.     

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.     

Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light.

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.     

DNA polymerase beta is required for efficient DNA strand break repair induced by methyl methanesulfonate but not by hydrogen peroxide

The most frequent DNA lesions in mammalian genomes are removed by the base excision repair (BER) via multiple pathways that involve the replacement of one or more nucleotides at the lesion site. The biological consequences of a BER defect are at present largely unknown. We report here that mouse cells defective in the main BER DNA polymerase β (Pol β) display a decreased rate of DNA single-strand breaks (ssb) rejoining after methyl methanesulfonate damage when compared with wild-type cells. In contrast, Pol β seems to be dispensable for hydrogen peroxide-induced DNA ssb repair, which is equally efficient in normal and defective cells. By using an in vitro repair assay on single abasic site-containing circular duplex molecules, we show that the long-patch BER is the predominant repair route in Pol β-null cell extract. Our results strongly suggest that the Pol β-mediated single nucleotide BER is the favorite pathway for repair of N-methylpurines while oxidation-induced ssb, likely arising from oxidized abasic sites, are the substrate for long-patch BER.     

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.     

Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.

Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds.     

Nonuniform distribution of DNA repair in chromatin after treatment with methyl methanesulfonate.

The distribution of methyl methanesulfonate induced DNA repair was measured in mouse mammary cell chromatin by digestion of "repair labeled" nuclei with micrococcal nuclease. The results indicate that there is a nonuniform distribution of DNA repair in chromatin. The chromatin fraction digested during the first 5 minutes of incubation with micrococcal nuclease appears to be a primary site of DNA repair after methyl methanesulfoante treatment. The observed nonuniform distribution of DNA repair in chromatin may be due to 1)a nonrandom alkylation of DNA in chromatin by methyl methanesulfonate or 2)areas in chromatin of increased accessibility for the repair enzymes to the DNA lesions.     

Identification and preliminary characterization of a Streptococcus sanguis fibrillar glycoprotein.

Cell surface fibrils could be released from Streptococcus sanguis 12 but not from strains 12na or N by freeze-thawing followed by brief homogenization. Fibrils were isolated from the homogenate by ultracentrifugation or ammonium sulfate precipitation. Electron microscopy demonstrated the presence of dense masses of aggregated fibrils in these preparations. Under nondenaturing conditions, no proteins were seen in polyacrylamide gel electrophoresis (PAGE). Sodium dodecyl sulfate (SDS)-PAGE analysis revealed a single band stained with Coomassie blue and periodic acid Schiff stain with a molecular weight in excess of 300,000. The protein has been given the name long-fibril protein (LFP). The molecule was susceptible to digestion with subtilisin, pronase, papain, and trypsin, but was unaffected by chymotrypsin or muramidases. Attempts to dissociate the protein into smaller subunits with urea, guanidine, sodium thiocyanate, and HCl were unsuccessful. Gel filtration on a column of Sephacryl S-400 in the presence of 2% SDS resulted in elution of the protein at the void volume. Antibody raised against the LFP excised from an SDS-PAGE gel reacted with long fibrils on the surface of strain 12 and with isolated fibrils by an immunogold labeling technique. Monoclonal antibody reactive with LFP in SDS-PAGE also reacted with fibrils present on the cell. Antisera raised against the fibrils inhibited adherence to saliva-coated hydroxyapatite.     

Identification and characterization of a DNase induced by Epstein-Barr virus.

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoyl-phorbol-13-acetate, induced a DNase activity in the Epstein-Barr virus-producer cell line P3HR-1. The elution patterns of the enzyme from DEAE-cellulose, phosphocellulose, and DNA-cellulose columns were different from virus-associated DNA polymerase activity. The partially purified activity could be neutralized to the extent of 90% by sera of patients with nasopharyngeal carcinoma. Purified immunoglobulin G from sera of nasopharyngeal carcinoma patients inhibited this enzyme and that obtained from superinfected Raji cells to the same extent. The partially purified enzyme preferred native DNA as a substrate over denatured DNA and 3'-terminally labeled activated calf thymus DNA. The activity was inhibited by high ionic strength. Phosphonoformic acid did not have any effect on this enzyme activity.     

Molecular cloning and characterization of a genetic region from Serratia marcescens involved in DNA repair

We report here the molecular isolation of a DNA fragment which encodes Tag-like activity from the Gram-negative bacterium Serratia marcescens. A recombinant plasmid encoding Tag-like activity was isolated from a S. marcescens plasmid gene library by complementation of an Escherichia coli tag mutant, which is deficient in 3-methyladenine DNA glycosylase I. The clone complements E. coli tag, recA, alkA, but not alkB, mutants for resistance to the DNA-damaging agent methyl methanesulphonate (MMS). The coding region of the Tag activity, initially isolated on a 6.5kb BamHI fragment, was defined to a 1.8kb BglII-SmaI fragment. Labelling of plasmid-encoded proteins using maxicells revealed that the 1.8kb fragment encodes two proteins of molecular weights 42,000 and 16,000. Data presented here suggest that the cloned fragment encodes a DNA repair protein(s) that has similar activity to the 3-methyladenine DNA glycosylase I of E. coli.     

Recruitment of HRDC domain of WRN and BLM to the sites of DNA damage induced by mitomycin C and methyl methanesulfonate

The HRDC (helicase and RNase D C-terminal) domain at the C-terminal of WRNp (Werner protein) (1150-1229 amino acids) and BLMp (Bloom protein) (1212-1292 amino acids) recognize laser microirradiation-induced DNA dsbs (double-strand breaks). However, their role in the recognition of DNA damage other than dsbs has not been reported. In this work, we show that HRDC domain of both the proteins can be recruited to the DNA damage induced by MMS (methyl methanesulfonate) and MMC (methyl mitomycin C). GFP (green fluorescent protein)-tagged HRDC domain produces distinct foci-like respective wild-types after DNA damage induced by the said agents and co-localize with γ-H2AX. However, in time course experiment, we observed that the foci of HRDC domain exist after 24 h of removal of the damaging agents, while the foci of full-length protein disappear completely. This indicates that the repair events are not completed by the presence of protein corresponding to only the HRDC domain. Consequently, cells overexpressing the HRDC domain fail to survive after DNA damage, as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Moreover, 24 h after removal of damaging agents, the extent of DNA damage is greater in cells overexpressing HRDC domain compared with corresponding wild-types, as observed by comet assay. Thus, our observations suggest that HRDC domain of both WRN and BLM can also recognize different types of DNA damages, but for the successful repair they fail to respond to subsequent repair events.     

Possible repair action of Vitamin C on DNA damage induced by methyl methanesulfonate, cyclophosphamide, FeSO4 and CuSO4 in mouse blood cells in vivo

Interaction between Vitamin C (VitC) and transition metals can induce the formation of reactive oxygen species (ROS). VitC may also act as an ROS scavenger and as a metal chelant. To examine these possibilities, we tested in vivo the effect of two doses of VitC (1 and 30 mg/kg of mouse body weight) on the genotoxicity of known mutagens and transition metals. We used the alkaline version of the comet assay to assess DNA damage in peripheral white blood cells of mice. Animals were orally given either water (control), cyclophosphamide (CP), methyl methanesulfonate (MMS), cupric sulfate or ferrous sulfate. A single treatment with each VitC dose was administered after treatment with the mutagens or the metal sulfates. Both doses of VitC enhanced DNA damage caused by the metal sulfates. DNA damage caused by MMS was significantly reduced by the lower dose, but not by the higher dose of VitC. For CP, neither post-treatment dose of VitC affected the DNA damage level. These results indicate a modulatory role of Vitamin C in the genotoxicity/repair effect of these compounds. Single treatment with either dose of VitC showed genotoxic effects after 24 h but not after 48 h, indicating repair. Double treatment with VitC (at 0 and 24 h) induced a cumulative genotoxic response at 48 h, more intense for the higher dose. The results suggest that VitC can be either genotoxic or a repair stimulant, since the alkaline version of the comet assay does not differentiate "effective" strand breaks from those generated as an intermediate step in excision repair (incomplete excision repair sites). Further data is needed to shed light upon the beneficial/noxious effects of VitC.     

Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination.

Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV. For cloning DNA that can repair a mutation in the spl pathway called spl-1, a library of EcoRI fragments of chromosomal DNA from B. subtilis 168 was constructed in integrative plasmid pJH101 and introduced by transformation into a mutant B. subtilis strain that carries both the uvrA42 and spl-1 mutations, and transformants whose spores exhibited UV resistance were selected by UV irradiation. With a combination of genetic and physical mapping techniques, the DNA responsible for the restoration of UV resistance was shown to be present on a 2.3-kb EcoRI-HindIII fragment that was mapped to a new locus in the metC-pyrD region of the B. subtilis chromosome immediately downstream from the pstI gene. The spl coding sequence was localized on the cloned fragment by analysis of in vitro-generated deletions and by nucleotide sequencing. The spl nucleotide sequence contains an open reading frame capable of encoding a 40-kDa polypeptide that shows regional amino acid sequence homology to DNA photolyases from a number of bacteria and fungi.     

Repair of apurinic/apyrimidinic sites by UV damage endonuclease; a repair protein for UV and oxidative damage.

UV damage endonuclease (UVDE) initiates a novel form of excision repair by introducing a nick imme-diately 5" to UV-induced cyclobutane pyrimidine dimers or 6-4 photoproducts. Here, we report that apurinic/apyrimidinic (AP) sites are also nicked by Neurospora crassa and Schizosaccharomyces pombe UVDE. UVDE introduces a nick immediately 5" to the AP site leaving a 3"-OH and a 5"-phosphate AP. Apyrimidinic sites are more effectively nicked by UVDE than apurinic sites. UVDE also possesses 3"-repair activities for AP sites nicked by AP lyase and for 3"-phosphoglycolate produced by bleomycin. The Uvde gene introduced into Escherichia coli cells lacking two types of AP endonuclease, Exo III and Endo IV, gave the host cells resistance to methylmethane sulfonate and t-butyl hydroperoxide. We identified two AP endonuclease activities in S.pombe cell extracts. Besides cyclobutane pyrimidine dimers and 6-4 photoproducts, N. crassa UVDE also nicks Dewar photoproducts. Thus, UVDE is able to repair both of the major forms of DNA damage in living organisms: UV-induced DNA lesions and AP sites.     

DNA single stranded gaps formed during DNA repair synthesis induced by methyl methanesulfonate are filled by sequential action of aphidicolin- and dideoxythymidine sensitive DNA polymerases in HeLa cells

DNA repair synthesis induced by methyl methanesulfonate in preconditioned HeLa cells in which DNA replicative synthesis had been highly suppressed was inhibited by aphidicolin (an inhibitor of DNA polymerases and ) and dideoxythymidine (ddThR, an inhibitor of DNA polymerase ). Incomplete repair patches sensitive to exonuclease III were accumulated in the presence of aphidicolin while not in the presence of ddThR. These patches were comopleted by the combined action of Klenow fragment and T4 DNA ligase, indicating that the single-stranded gaps were formed during the repair synthesis. Moreover, ddThR had little effect on the repair synthesis in the presence of aphidicolin. Thus, the results suggest that the single-stranded gaps may be sealed first by aphidicolin-sensitive polymerase followed by ddThR-sensitive DNA polymerase on the same site of the repair patch.Abbreviations ddThR dideoxythymidine - MMS methyl methanesulfonate - dNTP deoxynucleoside triphosphate     

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.