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Summary The DNA polymerase deficient mutantE. coli P3478polA1 is extremely sensitive to the lethal action of N-methyl-N-nitro-N-nitrosoguanidine (NG) and methyl-methanesulfonate (MMS). ThepolA1 mutant has an almost unaffected mutability induced by NG or MMS and shows reduced ability to propagate MMS-treated phage T7. NG and MMS induce marked breakdown of DNA and inhibit significantly DNA synthesis in thepolA1 mutant. The obtained results suggest the thepolA1 mutant is unable to repair single-strand breaks of DNA induced by monofunctional alkylating agents. The suggestion is confirmed by the demonstrated sensitivity of thepolA1 mutant to thymine starvation (TS).  相似文献   

3.
A polA1 exrA strain of Escherichia coli K-12 was constructed. It was found to be more sensitive to aerobic or anoxic X irradiation than were mutants containing either polA1 or exrA alone. The ability of polA1 exrA and related strains to repair X-ray-induced single-strand breaks in deoxyribonucleic acid DNA was examined. The polA1 strain was deficient in type II (buffer) repair but not in type III (growth medium-dependent) repair. The exrA strain was not deficient in type II repair but was deficient in type III repair (similar to rec strains). The double mutant polA1 exrA was deficient in both type II and type III repair. Thus, the increased X-ray sensitivity of the polA1 exrA double mutant was correlated with its decreased ability to repair X-ray-induced single-strand breaks in DNA. We have tested the hypothesis that polA rec double mutants are not viable because they lack the types II and III systems for the repair of DNA single-strand breaks. Since the polA1 exrA strain is viable and is deficient in both of these repair processes, this hypothesis seems not to be correct.  相似文献   

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Summary A new mutation affecting DNA polymerase I of Escherichia coli is described. Strains carrying mutation polA107 are similar to polA1 strains in their sensitivity to methyl methanesulphonate (MMS), thymine deprivation, their reduced ability to repair MMS treated phage and are unable to propagate a phage red - mutant.Like the polA1-recBC combination, polA107-recBC double mutants are inviable. However, in contrast to polA1 mutants, polA107 mutants grow almost normally in the presence of acridine orange. PolA107 bacteria are more sensitive to UV and X-ray irradiation than Pol+ strains but not as sensitive as polA1 strains. Following X-ray irradiation, DNA degradation in the polA107 strains is as extensive as in the polA1 strain. X-ray induced single-strand breaks, however, are repaired in the polA107 strain but not in the polA1 strain. Following UV irradiations in contrast to the polA1 strain, only low levels of DNA degradation were observed in the polA107 strain.Complementation for MMS or radiation resistance between the polA107 and polA1 mutations was not observed. In the following paper it is shown that the polA107 strain contains a normal level of DNA polymerizing activity but lacks the associated 5–3 exonucleolytic activity found in DNA polymerase I.  相似文献   

5.
Non-glucosylated, non-methylated phage T2 DNA was methylated in vitro with partially purified wild-type (dam+) or mutant (damh) T2 DNA adenine methylase. The radioactively labeled methyladenine-containing DNA was enzymatically degraded and the resulting oligonucleotides were separated according to chain length by DEAE-cellulose chromatography. Following “fingerprinting” by two-dimensional electrophoresis, we determined the sequence for various di-, tri- and tetranucleotides containing radioactive N6-methyldeoxyadenosine. From this analysis we conclude that both T2 dam+ and T2 damh contain the sequence 5′…G-mA -Py…3′.  相似文献   

6.
Summary F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30–200 times higher than the isogenic dam + strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.  相似文献   

7.
Summary Bacteria mutant in the dam (DNA adenine methylation) gene and in either recA or recB or recC genes are inviable (Virm- phenotype). From crosses between dam-3 bacteria and recA1 or recB21 recC22 strains, Vrm+ recombinants were recovered. Among these recombinants, Dam+ revertants were present which did not show the phenotypes normally associated with dam-3 bacteria. Three classes of indirectly suppressed strains (dam-3 genotype) were also recovered which showed alterations in the secondary phenotypes normally associated with dam-3 bacteria. These strains contained a second unlinked mutation in either mutL or mutS or sin. In addition, mutation in either sbcA or sbcB suppresses the Vrm- phenotype of dam-3 recB21 recC22 strains.  相似文献   

8.
Location of DNA methylation genes on the Escherichia coli K-12 genetic map   总被引:73,自引:0,他引:73  
Summary The genes responsible for DNA adenine methylation (dam) and DNA cytosine methylation (dcm) have been mapped on the E. coli K-12 genetic map. The dam gene is situated at min 65 and the gene order cysG-(trpS, dam)-aro B inferred. The dcm gene is located at min 37.5 and the gene order is supD-dcm-flaA1. In F merodiploids, the dam and dcm alleles are recessive.  相似文献   

9.
The wild-type (dam+) and mutant (damh) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam+ and damh enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 damh enzyme methylated λ DNA to a higher level than did the dam+ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 damh, but not T2 dam+ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam+ and damh were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam3 DNA (contains no N6-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 damh modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam3 DNA was partially methylated to the same level with T2 dam+ or T2 damh; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam+ enzyme methylates G-A-C sequences less efficiently than the T2 damh methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.  相似文献   

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Summary We have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam -). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD and dinF) were found to express higher levels of -galactosidase in dam - strains than in isogenic dam + strains. The attempted construction of dam - strains that were also mutant in one of several SOS genes indicated that the viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for dam - strain viability.  相似文献   

12.
《Biochimie》1987,69(5):539-541
The wild type glnS promoter contains a dam methylation site. In dam strains, the expression of glnS is enhanced 2.6-fold. A mutated form of the promoter has been isolated in which the dam methylation site is lost. Expression of this promoter is insensitive to dam methylation.  相似文献   

13.
Summary We have found that the cells possessing the polA6 mutation affecting DNA polymerase I are unable to accept another mutation (uvr502) leading to UV-sensitivity. The introduction of the polA12 mutation determining the synthesis of a temperature sensitive DNA polymerase I into the uvr502 mutant results in the temperature sensitivity of colony forming ability of the double mutant. These data show that the uvr502 derivatives lacking DNA polymerase I are inviable. Reversions to temperature resistance in the population of the double mutant uvr502 polA12 may occur because of reverse mutations at one of the mutated sites or because of mutations suppressing DNA polymerase I deficiency but not UV- or MMS-sensitivity of revertants. DNA and protein synthesis in uvr502 polA12 cells continues after a shift to 45°C with rates almost indistinguishable from those in single mutants or wild type cells. No differences in DNA degradation were observed during incubation of single and double mutants at 45°C. The single strand molecular weight distribution of parent DNA from the double mutant as well as that from wild type cells is not affected by the shift to 45°C and 3 hours incubation at this temperature. We suggest that DNA polymerase I and/or the product altered by the uvr502 mutation are required for some step(s) of discontinuous DNA replication nonessential for the formation of acid insoluble DNA. The DNA polymerase I and the uvr gene product seem to be able to substitute for each other in accomplishing this process.  相似文献   

14.
Summary E. coli strains carrying the dam-3 and dam-4 mutations resulting in reduced levels of 6-methyladenine in the DNA have been found to be more sensitive to base analogue mutagenesis than dam + strains. Mutagenesis by EMS was also found to be enhanced in dam strains. Dam mutants however were not found to be hypermutable by UV light. It is concluded that the dam strains are deficient in the correct repair of mispairing lesions. The data are consistent with the hypothesis that 6-methyladenine residues in the DNA are involved in strand discrimination during mismatch correction.  相似文献   

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The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N6-adenine) methyltransferases. T. pallidum Dam can be assigned to group α DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam+ phenotype).  相似文献   

17.
Summary The induction of prophage by ultraviolet light has been measured inE. coli K12 lysogenic cells deficient in DNA polymerase I. The efficiency of the induction process was greater inpolA1 polC(dnaE) double mutants incubated at the temperature that blocks DNA replication than inpolA + polC single mutants. Similarly, thepolA1 mutation sensitizedtif-promoted lysogenic induction in apolA1 tif strain at 42°. In strains bearing thepolA12 mutation, which growth normally at 30°, induction of the prophage occured after the shift to 42°. It is concluded that dissapearance of the DNA polymerase I activity leads to changes in DNA replication that are able, per se, to trigger the prophage induction process.  相似文献   

18.
Escherichia coli K12 uvrE is a mutator strain which is highly sensitive to ultraviolet (UV) radiation.In an attempt to determine the underlying molecular basis for the UV sensitivity, we have compared a mutant and an isogenic wild type strain with regard to several metabolic responses to 254-nm radiation. The introduction of single-strand breaks into intracellular DNA after irradiation is normal. However, the rate of excision of pyrimidine dimers as well as of DNA degradation and final rejoining of the strand breaks is lower in the mutant as compared to the repair proficient strain.These data suggest that the uvrE gene product may be involved in a reaction between the incision and excision steps in the excision repair process.  相似文献   

19.
Summary The formation and repair of double-strand breaks induced in DNA by MMS was studied in haploid wild type and MMS-sensitive rad6 mutant strains of Saccharomyces cerevisiae with the use of the neutral and alkaline sucrose sedimentation technique. A similar decrease in average molecular weight of double-stranded DNA from 5–6x108 to 1–0.7x108 daltons was observed following treatment with 0.5% MMS in wild type and mutant strains. Incubation of cells after MMS treatment in a fresh drug-free growing medium resulted in repair of double-strand breaks in the wild type strain, but only in the exponential phase of growth. No repair of double-strand breaks was found when cells of the wild type strain were synchronized in G-1 phase by treatment with factor, although DNA single-strand breaks were still efficiently repaired. Mutant rad6 which has a very low ability to repair MMS-induced single-strand breaks, did not repair double-strand breaks regardless of the phase of growth.These results suggest that (1) repair of double-strand breaks requires the ability for single-strand breaks repair, (2) rejoining of double-strand breaks requires the availability of two homologous DNA molecules, this strongly supports the recombinational model of DNA repair.  相似文献   

20.
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.  相似文献   

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