Effect of recF, recJ, recN, recO and ruv mutations on ultraviolet survival and genetic recombination in a recD strain of Escherichia coli K12

Summary DNA repair and recombination were investigated in a recD mutant of Escherichia coli which lacked the nuclease activity of the RecBCD enzyme. The resistance of this mutant to ultraviolet (UV) light was shown to be a function of recJ. A recD recJ double mutant was found to be more sensitive to UV radiation than a recB mutant, whereas recD and recJ single mutants were resistant. Recombination in conjugational crosses with Hfr donors was also reduced in recD recJ strains, but the effect was modest in comparison with the sensitivity to UV. Within certain limits, mutations in recF, recN, recO, lexA and ruv did not affect sensitivity to UV and recombination in a recD mutant any more than in a recD ⁺ strain. The possibility that recD and recJ provide overlapping activities, either of which can promote DNA repair and recombination in the absence of the other, is discussed.     

The effect of a drug-resistance factor on recombination and repair of DNA in Escherichia coli K12.

The presence in recipient strains of Escherichia coli K12 of the plasmid R46 greatly reduced the yield of recombinants from crosses with several Hfr strains and virtually abolished the formation of recombinants by PI transduction without, however, significantly affecting the transfer of the F prime from a strain carrying Fgal. The R46 plasmid had paradoxical effects on mutability: it appeared to enhance the yield of mutants following irradiation with ultraviolet ligh but it reduced the number of mutants detectable in unirradiated cultures. The effects of this plasmid on ultraviolet survival of the wild type and several mutants defective for recombination and repair have been measured and the results, in the main, confirm similar observation by Tweats et al. (1976). Not only is the survival of the strain habouring R46 greater than that of the parent strain in all the cases studied, but the survival of ultraviolet irradiated bacteriophage lambda is also greater.     

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12

Summary Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strain. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv ⁺ product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...     

Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12

The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.     

Thermoresistant revertants of an Escherichia coli strain carrying tif-1 and ruv mutations: non-suppressibility of ruv by sfi.

Spontaneous thermoresistant revertants were isolated from Tif1 Ruv- and Tif1 Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tif+ and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenyotype were detected at a higher frequency in the Tif1 Ruv- strains (65%) than in the Tif1 Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating lambda bio11 phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lex, LEXB, or zab mutations, were UV sensitive and were able to propagate lambda bio11 phage (group 5). The sfi mutation, which suppresses filamentation in the Tif1 and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant.     

A molecular model for conjugational recombination in Escherichia coli K12

Summary Conjugational recombination in Escherichia coli was investigated by measuring lacZ ⁺ product, -galactosidase, in crosses between lacZ mutants. Enzyme production in both Hfr and F-prime crosses was detected very soon after transfer of the donor lacZ allele. The level of enzyme activity was reduced by no more than two-fold when the recipient carried a recB mutation. With an F-prime donor, recombination appeared to be restricted largely to a short period immediately after transfer, with little evidence of recombination during subsequent exponential growth of the transconjugant cells. These observations are interpreted to suggest that recA dependent recombination is able to initiate with high efficiency at gaps present in the donor DNA before synthesis of a complementary strand is completed, and independently of recB function. A molecular model for conjugational recombination based on this idea is presented in terms of the known activities of recA and recBC products. Some of the predictions of the model are tested by analysing the recombinant genotypes produced in Hfr crosses with multiply marked strains.     

Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12.

The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA. Strains carrying null mutations in recG show reduced recombination and DNA repair. Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA. Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins. Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs. The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon. Multicopy priA+ plasmids increase substantially the recombination- and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains. The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA. It is concluded that the 3'-5' DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG.     

glnA mutations conferring resistance to methylammonium in Escherichia coli K12

Cells of Escherichia coli K12 were sensitive to 100 mM-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4Cl or glutamine. Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype. Mutants were isolated which were resistant to 100 mM-methylammonium, even when grown under nitrogen limitation. P1 bacteriophage transduction and F' complementation analysis revealed that the resistance-conferring mutations mapped either inside the glnA structural gene and/or elsewhere in the E. coli chromosome. Glutamine synthetase was purified from the wild-type and from some of the mutant strains. Strains carrying glnA-linked mutations that were solely responsible for the methylammonium-resistant phenotype yielded an altered enzyme, which was less active biosynthetically with either ammonium or methylammonium as substrate. Sensitivity to methylammonium appeared to be due to synthesis of gamma-glutamylmethylamide by glutamine synthetase, which was synthesized poorly, if at all, by mutants carrying an altered glutamine synthetase enzyme.     

Genetic recombination in Escherichia coli. IV. Isolation and characterization of recombination-deficiency mutants of Escherichia coli K12

19 independent recombination-deficient mutants were isolated. 7 carried mutations that mapped near or in the recB and recC genes between thyA and argA. 10 mutants carried mutations cotransducible with pheA and exhibited no complementation with recA in temporary zygotic diploids.