Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB.

The molecular mechanisms for the recF-dependent and recB-dependent pathways of postreplication repair were studied by sedimentation analysis of DNA from UV-irradiated Escherichia coli cells. When the ability to repair DNA daughter strand gaps was compared, uvrB recF cells showed a gross deficiency, whereas uvrB recB cells showed only a small deficiency. Nevertheless, the uvrB recF cells were able to perform some limited repair of daughter strand gaps compared with a "repairless" uvrB recA strain. The introduction of a recB mutation into the uvrB recF strain greatly increased its UV radiation sensitivity, yet decreased only slightly its ability to repair daughter strand gaps. Kinetic studies of DNA repair with alkaline and neutral sucrose gradients indicated that the accumulation of unrepaired daughter strand gaps led to the formation of low-molecular-weight DNA duplexes (i.e., DNA double-strand breaks were formed). The uvrB recF cells were able to regenerate high-molecular-weight DNA from these low-molecular-weight DNA duplexes, whereas the uvrB recF recB and uvrB recA cells were not. A model for the recB-dependent pathway of postreplication repair is presented.     

Role of the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB

In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells. The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells. Thus, the radB gene does not appear to be involved in the recF-dependent or recF recB-independent processes for the repair of DNA daughter-strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strand breaks.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

Role of the umuC gene in postreplication repair in UV-irradiated Escherichia coli K-12 uvrB

The role of the umuC gene product in postreplication repair was studied in UV-irradiated Escherichia coli K-12 uvrB cells. A mutation at umuC increased the UV radiation sensitivities of uvrB, uvrB recF, uvrB recB, and uvrB recF recB cells; it also increased the deficiencies in the repair of DNA daughter-strand gaps in these strains, but it did not affect the repair of DNA double-strand breaks that arose from unrepaired DNA daughter-strand gaps. We suggest that the umuC gene product is involved in a minor system for the repair of DNA daughter-strand gaps, possibly the repair of overlapping DNA daughter-strand gaps.     

Role of DNA polymerase I in postreplication repair: a reexamination with Escherichia coli delta polA.

Using strains of Escherichia coli K-12 that are deleted for the polA gene, we have reexamined the role of DNA polymerase I (encoded by polA) in postreplication repair after UV irradiation. The polA deletion (in contrast to the polA1 mutation) made uvrA cells very sensitive to UV radiation; the UV radiation sensitivity of a uvrA delta polA strain was about the same as that of a uvrA recF strain, a strain known to be grossly deficient in postreplication repair. The delta polA mutation interacted synergistically with a recF mutation in UV radiation sensitization, suggesting that the polA gene functions in pathways of postreplication repair that are largely independent of the recF gene. When compared to a uvrA strain, a uvrA delta polA strain was deficient in the repair of DNA daughter strand gaps, but not as deficient as a uvrA recF strain. Introduction of the delta polA mutation into uvrA recF cells made them deficient in the repair of DNA double-strand breaks after UV irradiation. The UV radiation sensitivity of a uvrA polA546(Ts) strain (defective in the 5'----3' exonuclease of DNA polymerase I) determined at the restrictive temperature was very close to that of a uvrA delta polA strain. These results suggest a major role for the 5'----3' exonuclease activity of DNA polymerase I in postreplication repair, in the repair of both DNA daughter strand gaps and double-strand breaks.     

Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed.     

recF-dependent and recF recB-independent DNA gap-filling repair processes transfer dimer-containing parental strands to daughter strands in Escherichia coli K-12 uvrB.

The processes for repairing DNA daughter-strand gaps were studied in UV-irradiated uvrB, uvrB recB, uvrB recF, and uvrB recB recF cells of Escherichia coli K-12. The dimer-containing parental DNA was found to be joined to daughter strands during postreplication repair in all four strains examined. Therefore, both the major (recF-dependent) and the minor (recF recB-independent) gap-filling processes repair DNA daughter-strand gaps by transferring parental strands into daughter strands.     

A minor pathway of postreplication repair in Escherichia coli is independent of the recB, recC and recF genes

After ultraviolet (UV) irradiation, an Escherichia coli K12 uvrB5 recB21 recF143 strain (SR1203) was able to perform a limited amount of postreplication repair when incubated in minimal growth medium (MM), but not if incubated in a rich growth medium. Similarly, this strain showed a higher survival after UV irradiation if plated on MM versus rich growth medium (i.e., it showed minimal medium recovery (MMR]. In fact, its survival after UV irradiation on rich growth medium was similar to that of a uvrB5 recA56 strain, which does not show MMR or postreplication repair. The results obtained with a uvrB5 recF332::Tn3 delta recBC strain and a uvrB5 recF332::Tn3 recB21 recC22 strain were similar to those obtained for strain SR1203, suggesting that the recB21 and recF143 alleles are not leaky in strain SR1203. The treatment of UV-irradiated uvrB5 recB21 recF143 and uvrB5 recF332::Tn3 delta recBC cells with rifampicin for 2 h had no effect on survival or the repair of DNA daughter-strand gaps. Therefore, a pathway of postreplication repair has been demonstrated that is constitutive in nature, is inhibited by postirradiation incubation in rich growth medium, and does not require the recB, recC and recF gene products, which control the major pathways of postreplication repair.     

Genetic and Physical Analysis of Plasmid Recombination in Recb Recc Sbcb and Recb Recc Sbca Escherichia Coli K-12 Mutants

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.     

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.     

Mechanism of sbcB-suppression of the recBC-deficiency in postreplication repair in UV-irradiated Escherichia coli K-12

Summary The mechanism by which an sbcB mutation suppresses the deficiency in postreplication repair shown by recB recC mutants of Escherichia coli was studied. The presence of an sbcB mutation in uvrA recB recC cells increased their resistance to UV radiation. This enhanced resistance was not due to a suppression of the minor deficiency in the repair of DNA daughter-strand gaps or to an inhibition of the production of DNA double-strand breaks in UV-irradiated uvrA recB recC cells; rather, the presence of an sbcB mutation, enabled uvrA recB recC cells to carry out the repair of DNA double-strand breaks. In the uvrA recB recC sbcB background, a mutation, at recF produced a huge sensitization to UV radiation, and it rendered cells deficient in the repair of both DNA daughter-strand gaps and DNA double-strand breaks. Thus, an additional sbcB mutation in uvrA recB recC cells restored their ability to perform the repair of DNA double-strand breaks, but the further addition of a recF mutation blocked this repair capacity.     

Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair.

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 sbcC 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.     

Effect of rec mutations on viability and processing of DNA damaged by methylmethane sulfonate in xth nth nfo cells of Escherichia coli

The role of recombination genes in the processing of DNA damaged by methlymethane sulfonate (MMS) was examined in an xth nth nfo strain of Escherichia coli K-12. Introduction of a recQ mutation did not increase the cell's sensitivity to MMS treatment. The presence of recF, recJ or recN mutation slightly increased the cell's sensitivity to MMS treatment. The introduction of recA or recB mutation into the cells led to inviability. Taken together, we suggest that replication of DNA containing apurinic/apyrimidinic (AP) sites in vivo will lead to the formation of secondary lesions. The repair of these secondary lesions requires the function of recA and recB genes, but does not appear to require recF, recJ, recQ or recN genes.     

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.     

The Genetic Dependence of Recombination in Recd Mutants of Escherichia Coli

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.     

Quantitation of the involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC genes in the repair of X-ray-induced DNA double-strand breaks in Escherichia coli

Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB). These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range. The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.     

Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA.

The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination. We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches. These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids. These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests. Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E. coli strains. The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background). Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed. There was no apparent bias in favor of the insertion or deletion of mutant sequences.     

Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.     

Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli.

The formation of recombinants in Hfr crosses was studied in Escherichia coli strains carrying combinations of genes known to affect recombination and DNA repair. Mutations in ruv and recG eliminate activities that have been shown to process Holliday junction intermediates by nuclease cleavage and/or branch migration. Strains carrying null mutations in both ruv and recG produce few recombinants in Hfr crosses and are extremely sensitive to UV light. The introduction of additional mutations in recF, recJ, recO, recQ, or recR is shown to increase the yield of recombinants by 6- to 20-fold via a mechanism that depends on recBC. The products of these genes have been linked with the initiation of recombination. We propose that mutation of recF, recJ, recO, recQ, or recR redirects recombination to events initiated by the RecBCD enzyme. The strains constructed were also tested for sensitivity to UV light. Addition of recF, recJ, recN, recO, recQ, or recR mutations had no effect on the survival of ruv recG strains. The implications of these findings are discussed in relation to molecular models for recombination and DNA repair that invoke different roles for the branch migration activities of the RuvAB and RecG proteins.     

Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli.

The stability of two ColE1-related plasmids (pRSF2124 and pMB9) was examined in strains of Escherichia coli multiply deficient in exonucleases I (sbcB), III (xthA), or V (recB recC). Any combination of exonuclease I, III, and V deficiency resulted in dramatically decreased stability of both pRSF2124 and pMB9. Inactivation of the RecF pathway by introducing either recF or recJ mutations to the recB recC subcB background resulted in nearly wild-type levels of stability for both plasmids. In contrast, the introduction of uvrD3 uvr-257, uvrE100, or recL152 into the recB21 recC22 sbcB15 strain did not affect plasmid stability. Furthermore, the amount of plasmid DNA recovered from pRSF2124 or pMB9 transformants of a xthA1 sbcB15 strain was strikingly reduced relative to that of a wild-type control. Taken together, these results suggest that some aspect of DNA repair is required for stable maintenance of ColE1-related plasmids in E. coli.