2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: Mismatches function as inducing signals?

Summary The EcoK restriction of unmodified phage is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU). 2AP treatment of bacteria affects specificially the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction. 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam ^- strains. We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs.     

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.     

Effect of proofreading and dam-instructed mismatch repair systems on N4-hydroxycytidine-induced mutagenesis

Summary The role of the proofreading (35 exonuclease) function of T4 DNA polymerase and the mismatch repair system ofE. coli on N⁴-hydroxycytidine (oh⁴Cyd)¹ induced mutagenesis was investigated. oh⁴Cyd-induced mutation is strongly suppressed when the proofreading activity increases as a result of the presence oftsCB87-antimutator polymerase or elevated temperature (43° C vs 30° C). Mutagenic activity of oh⁴Cyd, however, is little, if at all, affected by the presence of thetsLB56 mutator allele of T4 DNA polymerase with suppressed proofreading activity. This leads to the conclusion that oh⁴C nucleotides are not frequently removed by proofreading activity of wild-type T4 DNA polymerase. The number of mutations induced by oh⁴Cyd increases 3- to 5-fold due to damage of the genesmutS,mutL,uvrE, but notmutR.Dam ^- cells are more sensitive to, and hypermutable by, oh⁴Cyd in comparison withdam ⁺ cells. This is compatible with the notion that oh⁴C residues are recognised and excised by mismatch repair enzymes. The results indicate thath neither the proofreading function of T4 DNA polymerase, nor the mismatch repair enzymes, are responsible for the high specificity of oh⁴Cyd which causes ATGC transition.     

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

Summary Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages appears to be correlated with their undermethylation during growth indam ⁺ (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 methyldirected mismatch repair system participates in counterselection of GATC sequences in enterobacteriophages, we have studied the yield of bacteriophage X174 containing either 0, 1, or 2 GATC sequences, in wild type,dam, andmut (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 bacterialmutH ⁺ 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 andmutL andmutS bacteria whereas the effect is not significant inmutU bacteria, suggesting an interaction of the, helicase II with the MutH protein.However, indam ⁺ 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 inmutH ^– bacteria reveals that methylated GATC sequences are advantageous to the phage. These results suggest that the methyl-directed mismatch repair system, and in particular its MutH protein, may have participated in severe counterselection of GATC sequences from enterobacteriophages, presumably, by DNA cleavage or by interfering with DNA replication or packaging when GATC sequences are undermethylated. Coevolution of the Dam and MutH proteins could then account for the loss of GATC sequences from DNA of bacteriophages growing indam ⁺ hosts.     

Effect of bacterial host repair systems on the viability of hydroxylamine and methyl methanesulfonate treated T4 and lambda bacteriophages

Summary Survival of HA or MMS-treated T4 and lambda phages was estimated in bacterial cells differing in their ability to repair DNA. It has been found that the mismatch repair system of the bacterial host, which involvesmutSmutRmutLuvrE anddam loci, does not excise, or does so to only a limited extent, the nonpaired bases from DNA of HA or MMS-treated phages. Mutation inpolA, both in the polymerase as well as in the 53 exonuclease activity, have a small effect on survival of HA-treated phages, whereas mutation in the polymerase activity has a pronounced effect on survival of MMS-treated phages. There was a difference in the effect of polA mutations on survival of MMS-treated T4 and lambda phages; the survival of the former was less affected than the latter. Induction of SOS response has no effect on repair of HA and MMS-treated phages. Pretreatment of bacterial host (including theada ^- mutant) with low doses of alkylating agents increases the survival of MMS (but not HA)-treated phages; pretreatment of bacteria with HA has no effect on survival of HA-treated phages. Three lines of evidence: the different inactivation rates of MMS-treated T4 and lambda phages, variation in the effect ofpolA mutations on survival of T4 and lambda phages, and a different level of adaptive response inada ^- cells towards of MMS-treated T4 and lambda phages, suggest that the patterns of DNA methylation in T4 and lambda phages are different.     

Asymmetric repair of bacteriophage T7 heteroduplex DNA

Summary Heteroduplex DNA molecules were prepared in vitro using one strand of DNA carrying a point mutation and one strand of the corresponding wild-type DNA. The heteroduplex DNA was transfected into competent bacteria and the progeny genotypes in the resulting infective centers were determined. From the results were conclude that about 80% of all transfected DNA molecules are repaired before DNA replication starts. This fraction of repaired DNA is independent of the location of the mismatched nucleotide pair. However, mismatch correction occurs preferentially on the H strand of the heteroduplex DNA.The repair does not depend on a known phage coded function but requires the active bacterial genes mut U, mut H, mut S and probably mut L.     

Location of DNA methylation genes on the Escherichia coli K-12 genetic map

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.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.     

The role of DNA polymerase I and the rec system in survival of bacteria and bacteriophages damaged by the photodynamic action of acridine orange

Summary The effect of acridine orange (AO)-sensitized photodynamic treatment (PD) was studied in various repair-deficient mutants of Salmonella typhimurium and Escherichia coli. Bacteria of either species carrying mutations in the polA gene and hence deficient in the enzyme DNA polymerase I were significantly more sensitive to PD-killing than polA ⁺ parent bacteria or phenotypically POL⁺ revertants of the polA strains (selected on the basis of resistance to methyl methanesulphonate). It therefore appears that DNA polymerase I plays an important role in cellular recovery from PD treatment. E. coli carrying a mutation in the recA gene was also more sensitive to PD-treatment than its parent strain, as was S. typhimurium carrying a mutation of the recA type. In S. typhimurium the rec mutant was somewhat less sensitive to PD-killing than the pol mutant even although it is much more sensitive to ultraviolet killing. E. coli strains with mutations in the recB and recC genes were intermediate in PD sensitivity between the recA and the parent strain. S. typhimurium and E. coli bacteria with mutations in the polA and recA genes showed reduced ability to host-cell reactivate PD-damaged bacteriophages ES 18 and c1, indicating that the polA ⁺ and recA ⁺ gene products also contribute to repair of bacteriophages damaged by PD treatment. It is suggested that the recombinational repair process is less important for recovery from PD than for recovery from UV, and that the primary contribution of the rec genes to recovery from PD may be in repair of single-strand gaps by repair resynthesis.     

The adaptive response of E.coli to low levels of alkylating agent: the role of polA in killing adaptation.

Summary In an attempt to characterise which gene products may be involved in the repair system induced in E. coli by growth on low levels of alkylating agent (the adaptive response) we have analysed mutants deficient in other known pathways of DNA repair for the ability to adapt to MNNG. Adaptive resistance to the killing effects of MNNG seems to require a functional DNA polymerase I whereas resistance to the mutagenic effects can occur in polymerase I deficient strains; similarly killing adaptation could not be observed in a dam3 mutant, which was nonetheless able to show mutational adaptation. These results suggest that these two parts of the adaptive response must, at least to some extent, be separable. Both adaptive responses can be seen in the absence of uvrD ⁺ uvrE ⁺-dependent mismatch repair, DNA polymerase II activity, or recF-mediated recombination and they are not affected by decreased levels of adenyl cyclase. The data presented support our earlier conclusion that adaptive resistance to the killing and mutagenic effect of MNNG is the result of previously uncharacterised repair pathways.