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M Radman

Publications and source records attributed to M Radman.

At least 37 records · Page 2Linked to original sources
PubMed1991

Mismatch-stimulated plasmid integration in yeast.

A single base pair mismatch (G:T or A:C) in the CYC1 gene of the integrative plasmid pAB218 stimulates up to a five-fold integration into the yeast chromosome. Analysis of chromosomal sites of plasmid integration suggests that the mismatch-stimulated integration is not targeted as would be expected if crossovers, localised in the region of the mismatch, were a necessary step in mismatch repair. Instead, the observed mismatch-stimulated plasmid integration could be due to potentially recombinogenic structures formed during mismatch repair, such as single-stranded gaps or denatured DNA regions extending around the plasmid molecule.

Base Composition↗
PubMed1991

Counterselection of GATC sequences in enterobacteriophages by the components of the methyl-directed mismatch repair system.

Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages appears to be correlated with their undermethylation during growth in dam+ (GATC ade-methylase) bacteria. This observation is corroborated by the sequence analysis showing no evidence for site-specific mutagenicity of 6meAde. The MutH protein of the methyl-directed mismatch repair system recognizes and cleaves the undermethylated GATC sequences in the course of mismatch repair. To enquire whether the MutH function of the methyl-directed mismatch repair system participates in counterselection of GATC sequences in enterobacteriophages, we have studied the yield of bacteriophage phi X174 containing either 0, 1, or 2 GATC sequences, in wild type, dam, and mut (H, L, S, U) Escherichia coli. Following transfection with unmethylated DNA containing two GATC sequences, a net decrease in the yield of infective particles was observed in all bacterial mutH+ dam- strains, whereas no detectable decrease was observed in bacteria infected by DNA without GATC sequence. This effect of the MutH function is maximum in wild type and mutL and mutS bacteria whereas the effect is not significant in mutU bacteria, suggesting an interaction of the helicase II with the MutH protein. However, in dam+ bacteria, the presence of GATC sequences leads to an increased yield of infective particles. The effect of GATC sequence and its Dam methylation system on phage yield in mutH- bacteria reveals that methylated GATC sequences are advantageous to the phage.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Avoidance of inter-repeat recombination by sequence divergence and a mechanism of neutral evolution.

Eucaryotic genomes are loaded with diverse repeated sequences and are therefore threatened by rearrangements via inter-repeat crossovers and by gene-inactivating conversions between genes and their inactive pseudogenes. Such repeated DNA sequences are usually diverged and polymorphic. Sequence divergence by well-spread point mutations is a potent inhibitor of homologous recombination due to the loss of recombination initiation sites and to the editing of recombinational intermediates by the mismatch repair system. Evidence is reviewed suggesting that a germ line process can identify duplicated sequences by homologous pairing, modify them by methylation and mutate by C----T transitions. Since this process requires a minimum contiguous homology that is larger than the average exon size, it is proposed that fragmentation by intron inserts protects the coding sequences from inactivation by homologous interactions with their pseudogene sequences.

Animals↗
PubMed1991

Interspecific recombination between Escherichia coli and Salmonella typhimurium occurs by the RecABCD pathway.

Interspecific recombination in conjugation between Escherichia coli and Salmonella typhimurium is several orders of magnitude lower than intraspecies recombination and is dependent on the RecA function. This low efficiency is due to a 20% divergence in the DNA sequence. The methyl-directed (mut H,L,S dependent) mismatch repair system appears to control the fidelity of homologous recombination; inactivating one of the Mut functions increases the interspecies recombination at least by 10(3)-fold. The interspecific recombination in mutS or mutL mutants is only approximately 10-fold lower than recombination in homospecific crosses as found after correction for the efficiency of mating and DNA transfer by zygotic induction experiments. The interspecific recombination is dependent on the RecABCD pathway: it was abolished in a recA mutant and decreased approximately 10(3)-fold in a recC mutant.

Conjugation, Genetic↗
PubMed1991

Control of large chromosomal duplications in Escherichia coli by the mismatch repair system.

Excessive recombination between repeated, interspersed, and diverged DNA sequences is a potential source of genomic instability. We have investigated the possibility that a mechanism exists to suppress genetic exchange between these quasi-homologous (homeologous) sequences. We examined the role of the general mismatch repair system of Escherichia coli because previous work has shown that the mismatch repair pathway functions as a barrier to interspecies recombination between E. coli and Salmonella typhimurium. The formation of large duplications by homeologous recombination in E. coli was increased some tenfold by mutations in the mutL and mutS genes that encode the mismatch recognition proteins. These findings indicate that the mismatch recognition proteins act to prevent excessive intrachromosomal exchanges. We conclude that mismatch repair proteins serve as general controllers of the fidelity of genetic inheritance, acting to suppress chromosomal rearrangements as well as point mutations.

Chromosomes, Bacterial↗
PubMed1990

Some restriction endonucleases tolerate single mismatches of the pyrimidine.purine type.

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.

Bacteriophages↗
PubMed1990

Genotoxic potency of monofunctional alkylating agents in E. coli: comparison with carcinogenic potency in rodents.

A quantitative correlation between carcinogenicity and genotoxicity was investigated by a comparison between the carcinogenic potency in rodents and the mutagenic (M), recombinogenic (R) and SOS-inducing (I) potencies in a bacterial test (E. coli multitest) for 9 monofunctional alkylating agents: N-nitroso-N-methylurethane, N-nitroso-N-ethylurea, epichlorohydrin, N-nitroso-N-methylurea, N-nitroso-N-methyl-N'-nitroguanidine, methyl methanesulfonate, diethylsulfate, dimethylsulfate, ethyl methanesulfonate. A significant positive correlation between the carcinogenic potency and the product of the mutagenic and recombinogenic potencies was found for all tested compounds. Thus, the E. coli multitest may be used as a simple test to search for correlations between carcinogenicity and genotoxicity of DNA-damaging agents.

Alkylating Agents↗
PubMed1990

DNA mismatch repair in Xenopus egg extracts: repair efficiency and DNA repair synthesis for all single base-pair mismatches.

Repair of all 12 single base-pair mismatches by Xenopus egg extracts was measured by a physical assay with a sequence containing four overlapping restriction sites. The heteroduplex substrates, derivatives of M13 phage DNA, differed in sequence at the mismatch position only and permitted measurement of repair to both strands. The efficiency of repair varied about 4-fold between the most and least effectively repaired mismatches. Repair was most active with C/A and T/C mismatches but the efficiency varied depending on the orientation of the mismatch. Mismatch-specific DNA repair synthesis was also observed but the extent of repair was not always predictive of the extent of synthesis, suggesting the presence of different repair systems or different modes of mismatch recognition.

Animals↗
PubMed1989

The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants.

The requirement for DNA sequence homology in generalized genetic recombination is greatly relaxed in bacterial mutL, mutS and mutH mutants deficient in mismatch repair. In such mutants, intergeneric recombination occurs efficiently between Escherichia coli and Salmonella typhimurium, which are approximately 20% divergent in DNA sequence. This finding has implications for speciation, for regulating recombination between diverged repeated sequences, and for hitherto difficult interspecies hybridizations.

Alleles↗
PubMed1989

The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors.

Escherichia coli mutator mutD5 is the most potent mutator known. The mutD5 mutation resides in the dnaQ gene encoding the proofreading exonuclease of DNA polymerase III holoenzyme. It has recently been shown that the extreme mutability of this strain results, in addition to a proofreading defect, from a defect in mutH, L, S-encoded postreplicational DNA mismatch repair. The following measurements of the mismatch-repair capacity of mutD5 cells demonstrate that this mismatch-repair defect is not structural, but transient. mutD5 cells in early log phase are as deficient in mismatch repair as mutL cells, but they become as proficient as wild-type cells in late log phase. Second, arrest of chromosomal replication in a mutD5-dnaA(Ts) strain at a nonpermissive temperature restores mismatch repair, even from the early log phase of growth. Third, transformation of mutD5 strains with multicopy plasmids expressing the mutH or mutL gene restores mismatch repair, even in rapidly growing cells. These observations suggest that the mismatch-repair deficiency of mutD strains results from a saturation of the mutHLS-mismatch-repair system by an excess of primary DNA replication errors due to the proofreading defect.

Base Sequence↗
PubMed1989

Genetic requirements for hyper-recombination by very short patch mismatch repair: involvement of Escherichia coli DNA polymerase I.

It has been established that very short patch (VSP) mismatch repair, depending in Escherichia coli on MutL, MutS and Dcm functions, is responsible for the hyper-recombinogenic effect of a class of genetic markers. We show that VSP repair requires the presence of the complete DNA polymerase I enzyme. The absence of endonuclease activities involved in the repair of base-loss sites, Nth, Nfo and Xth, does not affect VSP repair. Implications for the mechanism of the VSP repair are discussed.

Base Composition↗
PubMed1989

Mismatch repair involving localized DNA synthesis in extracts of Xenopus eggs.

Repair of heteroduplex DNA containing G.T or A.C mismatches or containing two tandem unpaired bases occurred in vitro with Xenopus egg extracts as detected by a physical assay. The repair was accompanied by a mismatch-stimulated and mismatch-localized DNA synthesis. Repaired molecules, separated from unrepaired molecules, showed a 20- to 100-fold increase in DNA synthesis in the region of the mismatch compared to regions distant from the mismatch. The remaining unrepaired heteroduplex DNA included molecules that also displayed mismatch-stimulated DNA synthesis in the mismatch-proximal regions. These may represent intermediates in the repair process. The patterns of DNA synthesis suggest that repair begins at some distance from the mismatch and that as much as 1 kilobase or more can be involved in the mismatch-stimulated synthesis.

Animals↗
PubMed1989

Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I.

The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process. The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type. The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type. The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation. The mutator activity therefore appears to reflect constitutive SOS induction. Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome. The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele. This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria. An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair. Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present.

DNA Polymerase I↗
PubMed1989

Saturation of mismatch repair in the mutD5 mutator strain of Escherichia coli.

The mutD (dnaQ) gene of Escherichia coli codes for the proofreading activity of DNA polymerase III. The very strong mutator phenotype of mutD5 strains seems to indicate that their postreplicational mismatch repair activity is also impaired. We show that the mismatch repair system of mutD5 strains is functional but saturated, presumably by the excess of DNA replication errors, since it is recovered by inhibiting chromosomal DNA replication. This recovery depends on de novo protein synthesis.

DNA Polymerase III↗
PubMed1989

Mismatch repair and the fidelity of genetic recombination.

Two modes of mismatch repair are known to operate in bacteria: long-patch mismatch repair and very short patch mismatch repair. Very short patch mismatch repair systems act on a specific mismatch by conserving only one base pair. Therefore, when very short patch mismatch repair acts on heteroduplex recombination intermediates, it hyper-recombines specific markers by creating patchwork sequences, i.e., apparent multiple exchange events, on the repaired strand. Long-patch mismatch repair is antirecombinagenic, apparently by decomposing heteroduplex DNA or aborting its formation whenever well-recognized mismatches are formed by strand exchange between nonidentical parental sequences. It is postulated here that mismatch-stimulated antirecombination by long-patch mismatch repair is a "proofreading" system assuring high fidelity of homologous recombination. This accounts for chromosomal stability in eucaryotes (i.e., the rare occurrence of chromosomal aberrations and mitotic recombination versus the high frequency of precise sister chromatid exchange), suggests a role for diverged repetitive and other noncoding sequences as chromosomal antirecombination elements, and provides a molecular mechanism for speciation without the necessity of geographical separation.

Biological Evolution↗
PubMed1988

Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA.

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.

Base Sequence↗
PubMed1987

Mismatch repair and recombination in E. coli.

The involvement of the E. coli methyl-directed and very short patch (vsp) mismatch repair systems in bacteriophage lambda recombination has been studied. Genetic crosses and heteroduplex transfection experiments were performed using lambda phages with sequenced mutations in the cl gene. The results indicate that methyl-directed repair does operate during bacteriophage lambda recombination but generally does not contribute to the formation of recombinants involving close markers. Vsp repair apparently acts during bacteriophage lambda recombination to produce recombinants involving close markers because its action does not involve extensive excision tracts. Marker-specific hyperrecombination and the apparent clustering of genetic exchanges in bacteriophage lambda recombination can be accounted for by the action of the vsp repair system.

Bacteriophage lambda↗