Fiona E. Benson

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

SummaryMutation 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).

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

SummaryRecombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.

Genetic Analysis of Conjugational Recombination in Escherichia coli K12 Strains Deficient in RecBCD Enzyme

Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme. The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations. The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.

Genetic analysis and molecular cloning of the Escherichia coli ruv gene.

SummaryThe genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(ApRlac)strains carrying F42lac+ established that ruv is transcribed in a counterclockwise direction. Recombinant λ phages able to restore UV resistance to ruv mutants were identified, and the ruv+ region was subcloned into a low copy number plasmid. The ruv+ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

SummaryGenetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec+sbc+ strains, depending on the plasmid used. Recombinant plasmids carrying ruv+ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA+strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.

Resolution of Holliday Junctions by the E. coli RuvC Protein

During genetic recombination, intermediates are formed in which two recombining DNA molecules are linked by a Holliday junction (Holliday 1964). There are two general questions regarding the mechanism by which Holliday junctions are resolved to form recombinant products: first, how is the junction recognized, and second, what are the biochemical mechanisms of the cleavage and ligation reactions. The recent identification of a Holliday junction-specific endonuclease from E. coli allows a detailed investigation of these problems and provides new insight into the mechanics of the late steps of genetic recombination.