We describe a transposon insertion that reduces the efficiency of homologous recombination and DNA repair in Escherichia coli. The insertion, rec-258, was located between pyrE and dgo at min 82.1 on the current linkage map. On the basis of linkage to pyrE and complementation studies with the cloned rec+ gene, rec-258 was identified as an allele of the recG locus first reported by Storm et al. (P. K. Storm, W. P. M. Hoekstra, P. G. De Haan, and C. Verhoef, Mutat. Res. 13:9-17, 1971). The recG258 mutation confers sensitivity to mitomycin C and UV light and a 3-to 10-fold deficiency in conjugational recombination in wild-type, recB recC sbcA, and recB recC sbcB sbeC genetic backgrounds. It does not appear to affect plasmid recombination in the wild-type. A recG258 single mutant is also sensitive to ionizing radiation. The SOS response is induced normally, although the basal level of expression is elevated two-to threefold. Further genetic studies revealed that recB recG and recG recJ double mutants are much more sensitive to UV light than the respective single mutants in each case. However, no synergistic interactions were discovered between recG258 and mutations in recF, recN, or recQ. It is concluded that recG does not fall within any of the accepted groups of genes that affect recombination and DNA repair.Genetic studies have so far linked some 16 genes (recA, recB, recCs recD, recE, recF, recJ, recN, recO, recQ, recR, ruvA, ruvB, ruvC, sbcB, sbcC) with the process of homologous reconibination in Escherichia coli K-12. For historical reasons, these have been defined as acting in the RecBCD, RecE, or kecF pathway of recombination depending on whether they are essential for the formation of recombinants in conjugational crosses with wild-type, recB recC sbcA, or recB recC sbcB sbcC strains (9). The only gene common to all three pathways is recA, which is indispensable for recombination (9) except in certain plasmid crosses with recBC sbcA strains (29,45). However, it has become clear that the genetic requirements for recombination vary with the type of cross conducted (10, 17), the DNA substrates presented (29,38), and the product assayed (4, 23). There is also a certain amount of redundancy. Thus, although recD, recJ, and recN single mutants are reasonably proficient in conjugational recombination, recD recJ double mutants have a reduced capacity (25, 28) and recD recJ recN triple mutants are quite deficient (19a). Presumably, the products of these three genes provide alternative routes to the formation of recombinant progeny. For these reasons, the idea of genes acting within disdrete pathways, with the RecBCD pathway predominating in the wild type (6,7,43), has become increasingly untenable (33).Nevertheless, the recB recC sbcB sbcC genotype (abbreviated hereafter to recBC sbcBC) used to define the RecF pathway has provided a remarkably fruitful genetic background for the identification of genes involved with recombination, since it was first used for this purpose by Horii and Clark (11). in a recent study, ...

The rescue of replication forks stalled on the template DNA was investigated using an assay for synthetic lethality that provides a visual readout of cell viability and permits investigation of why certain mutations are lethal when combined. The results presented show that RecA and other recombination proteins are often engaged during replication because RecA is present and provokes recombination rather than because recombination is necessary. This occurs particularly frequently in cells lacking the helicase activities of Rep and PriA. We propose that these two proteins normally limit the loading of RecA on ssDNA regions exposed on the leading strand template of damaged forks, and do so by unwinding the nascent lagging strand, thus facilitating reannealing of the parental strands. Gap closure followed by loading of the DnaB replicative helicase enables synthesis of the leading strand to continue. Without either activity, RecA loads more frequently on the DNA and drives fork reversal, which creates a chickenfoot structure and a requirement for other recombination proteins to re-establish a viable fork. The assay also reveals that stalled transcription complexes are common impediments to fork progression, and that damaged forks often reverse independently of RecA.[Keywords: DNA repair; RuvABC; RecBCD, RecFOR; Holliday junction] Supplemental material is available at http://www.genesdev.org.

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.