sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.     

sbcB15 And DeltasbcB mutations activate two types of recf recombination pathways in Escherichia coli

Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and DeltasbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and DeltasbcB mutations on DNA repair after UV and gamma irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than DeltasbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as DeltasbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC DeltasbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.     

Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12.

In Escherichia coli K-12, sbcB/xonA is the structural gene for exonuclease I, an enzyme that hydrolyzes single-stranded DNA to mononucleotides in the 3'-to-5' direction. This enzyme has been implicated in the DNA repair and recombination pathways mediated by the recB and recC gene products (exonuclease V). We have cloned several sbcB/xonA mutant alleles in bacterial plasmids and have partially characterized the cloned genes and their protein products. Two of the mutations (xonA2 and xonA6) retain no detectable exonucleolytic activity on single-stranded DNA. The xonA6 allele was shown to harbor an insertion of an IS30-related genetic element near the 3' end of the gene. Two other mutations, sbcB15 and xonA8, exhibited significantly reduced levels of exonuclease I activity as compared to the cloned wild-type gene. A correlation was observed between levels of exonuclease I activity and the ability of the sbcB/xonA mutations to suppress UV sensitivity in recB and recC strains. Also, recombinant plasmids bearing either the sbcB15 or xonA6 allele exhibited a high degree of instability during growth of their bacterial hosts. The results suggest that the sbcB/xonA gene product is a bi- or multifunctional protein that interacts with single-stranded DNA and possibly with other proteins in the suppression of genetic recombination and DNA-repair deficiencies in recB and recC mutants.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

Escherichia Coli Xona (Sbcb) Mutants Enhance Illegitimate Recombination

Mutations of Escherichia coli K-12 were isolated that increase the frequency of deletion formation. Three of these mutations map to the gene sbcB at 43.5 min on the E. coli chromosome. Two types of mutations at sbcB have been previously defined: sbcB-type that suppress both the UV sensitivity and recombination deficiency of recBC mutants, and xonA-type that suppress only the UV sensitivity. Both types are defective for production of exonuclease I activity. The mutations isolated here were similar to xonA alleles of sbcB because they suppressed the UV sensitivity of recBC mutants but did not restore recombination proficiency. Indeed, two previously characterized xonA alleles were shown to increase the frequency of deletion formation, although an sbcB allele did not. This result demonstrates that loss of exonuclease I activity is not sufficient to confer a high deletion phenotype, rather, the product of the sbcB gene possesses some other function that is important for deletion formation. Because deletion formation in this system is recA independent and does not require extensive DNA homology, these mutations affect a pathway of illegitimate recombination.     

Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: effects of mutations influencing genetic recombination and DNA metabolism.

Repair of cross-linked DNA was studied in Escherichia coli strains carrying mutations affecting DNA metabolism. In wild-type cells, DNA strands cut during cross-link removal were rejoined during a subsequent incubation into high-molecular-weight molecules. This rejoining was dependent on gene products involved in genetic recombination. A close correlation was found relating recombination proficiency, the rate of strand rejoining, and formation of viable progeny after DNA cross-linking by treatment with psoralen and light. Wild-type cells and other mutants which were Rec+ (sbcB, recL, recL sbcB, recB recC sbcA, recB recC sbcB, xthA1, and xthA11) rejoined cut DNA strands at a rate of 0.8 +/- 0.1 min -1 at 37 degrees C and survived 53 to 71 cross-links per chromosome. recB, recC, recB recC, recF, or polA strains showed reduced rates of strand rejoining and survived 4 to 13 cross-links per chromosome. Recombination-deficient strains (recA, recB recC sbcB recF, recB recL) and lexA failed to rejoin DNA strands after crosslink removal and were unable to form colonies after treatments producing as few as one to two cross-links per chromosome. Strand rejoining occurred normally in cells with mutations affecting DNA replication (dnaA, danB, dnaG, and dnaE) under both permissive and nonpermissive conditions for chromosome replication. In a polA polB dnaE strain strand rejoining occurred at 32 degree C but not at 42 degree C, indicating that some DNA synthesis was required for formation of intact recombinant molecules.     

RecF recombination pathway in Escherichia coli cells lacking RecQ, UvrD and HelD helicases

In recBCD sbcB sbcC(D) mutants of Escherichia coli homologous recombination proceeds via RecF pathway, which is thought to require RecQ, UvrD and HelD helicases at its initial stage. It was previously suggested that depletion of all three helicases totally abolishes the RecF pathway. The present study (re)examines the roles of these helicases in transductional recombination, and in recombinational repair of UV-induced DNA damage in the RecF pathway. The study has employed the ΔrecBCD ΔsbcB sbcC201 and ΔrecBCD sbcB15 sbcC201 strains, carrying combinations of mutations in recQ, uvrD, and helD genes. We show that in ΔrecBCD ΔsbcB sbcC201 strains, recombination requires exclusively the RecQ helicase. In ΔrecBCD sbcB15 sbcC201 strains, RecQ may be partially substituted by UvrD helicase. The HelD helicase is dispensable for recombination in both backgrounds. Our results also suggest that significant portion of recombination events in the RecF pathway is independent of RecQ, UvrD and HelD. These events are initiated either by RecJ nuclease alone or by RecJ nuclease associated with an unknown helicase. Inactivation of exonuclease VII by a xseA mutation further decreases the requirement for helicase activity in the RecF pathway. We suggest that elimination of nucleases acting on 3' single-strand DNA ends reduces the necessity for helicases in initiation of recombination.     

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.     

Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair

Summary A new recombination gene called recR has been identified and located near dnaZ at minute 11 on the current linkage map of Escherichia coli. The gene was detected after transposon mutagenesis of a recB sbcB sbcC strain and screening for insertion mutants that had a reduced efficiency of recombination in Hfr crosses. The recR insertions obtained conferred a recombination deficient and extremely UV sensitive phenotype in both recB recC sbcA and recB recC sbcB sbcC genetic backgrounds. recR derivatives of recBC ⁺ sbc ⁺ strains were proficient in conjugational and transductional recombination but deficient in plasmid recombination and sensitive to UV light. Strains carrying recR insertions combined with mutations uvrA and other rec genes revealed that the gene is involved in a recombinational process of DNA repair that relies also on recF and recO, and possibly recJ, but which is independent of recB, recC and recD. The properties of two other insertions, one located near pyrE and the other near guaA, are discussed in relation to their proximity to recG and xse (the gene for exonuclease VII), respectively.     

Effect of the<Emphasis Type="Italic"> recU</Emphasis> suppressors<Emphasis Type="Italic"> sms</Emphasis> and<Emphasis Type="Italic"> subA</Emphasis> on DNA repair and homologous recombination in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

In Bacillus subtilis, mutant alleles of the genes sms and subA partially suppress the recombination phenotype of recU cells. When present in an otherwise Rec(+) strain, Delta sms and Delta subA alleles render cells slightly sensitive to DNA-damaging agents, and impair chromosomal transformation (3- to 10-fold reduction), but do not affect plasmid transformation (less than 1.5-fold reduction). The Delta sms and Delta subA alleles were introduced into rec-deficient strains representative of the epistatic groups alpha (recF strain), beta (addA addB), gamma (recH), epsilon (recB, Delta recU and recD strains) and zeta (Delta recS). Both the Delta sms and Delta subA mutations were found to increase sensitivity to DNA-damaging agents in recF, Delta recS and addAB cells. In contrast, the Delta sms mutations decreased the sensitivity of recB, Delta recU, recD and recH cells, and the Delta subA mutation decreased the sensitivity of recB and Delta recU cells to DNA-damaging agents. Functions classified within the epistatic groups alpha, epsilon and zeta are required for intramolecular recombination, measured as plasmid transformation. The Delta sms and Delta subA mutations, which partially suppressed the recombinational repair phenotype of mutants for functions within epistatic group epsilon, enhanced plasmid transformation of recU (recB, recD) and recS cells by 10- to 20-fold. In the absence of the proteins Sms and SubA, the recombination machinery is apparently redirected towards (an) alternative pathway(s). Furthermore, the shared ability of the Delta sms and Delta subA mutations to act as indirect suppressors of recB, recU and recD mutations supports the classification of the recBUD genes within epistatic group epsilon.     

Effects of single-strand DNases ExoI, RecJ, ExoVII, and SbcCD on homologous recombination of recBCD+ strains of Escherichia coli and roles of SbcB15 and XonA2 ExoI mutant enzymes

To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD⁺ background, we studied 31 strains with all combinations of null alleles of exonuclease I (Δxon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD Δxon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon⁺), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3′ DNA overhangs, giving protection against Xon⁺. In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD⁺ cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.     

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.     

Genetic Analysis of Mutations Indirectly Suppressing recB and recC Mutations

Mutations in sbcB inactivate exonuclease I and suppress the UV-sensitive, mitomycin-sensitive, recombination-deficient phenotypes associated with recB and recC mutations. Mapping experiments have located sbcB about 0.4 minutes from the his operon at 38.0 on the standard map of E. coli. This places sbcB between supD and his. A four-point cross shows that sbcB lies between P2 attH and his. P2 eduction deleting the his operon beginning with P2 attH also deletes sbcB and produces the expected exonuclease I deficiency and suppression of recB(-). The occurrence of chemical-mutagen-induced and spontaneous mutations indirectly suppressing recB(-) and recC(-) is examined. Three lines of strains produce only sbcA mutations while only sbcB mutations occur in a fourth line. Explanations for this behavior are proposed in light of the ability of the first three lines to express sbcB mutations which they inherit by transduction.     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.     

Characteristics of Some Multiply Recombination-Deficient Strains of Escherichia coli

Strains of Escherichia coli have been made carrying lesions in more than one gene determining recombination. The following genotypes were constructed and verified: recC22 recB21 recA(+), recC22 recB21 recA13, recC22 recB(+)recA13, and recC(+)recB21 recA13. All multiple rec(-) strains carrying recA13 were similar to AB2463, which carries recA13 alone, in their UV sensitivities, recombination deficiencies, and inabilities to induce lambda phage in a lysogen. However, whereas AB2463 shows a high rate of ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown, the multiple rec(-) strains showed the low level characteristic of strains carrying recC22 or recB21 alone. The strain carrying both recC22 and recB21 was similar in all properties to the single mutants, suggesting that both gene products act in the same part of the recombination and UV repair pathways. It is concluded that in a Rec(+) strain, the recA(+) product acts to inhibit DNA breakdown determined by the recC(+) and recB(+) products.     

Plasmidic recombination in Escherichia coli K-12: the role of recF gene function

Interplasmidic and intraplasmidic recombination proficiencies were determined in E. coli bacterial strains carrying rec mutations. Our results defined the role of recF gene function, recB, recC, and sbcB gene products (exonuclease V and exonuclease I) in plasmidic recombination in wild-type E. coli cells and in cells in which the recE recombination pathway is activated. RecF gene function is required for interplasmidic recombination regardless of the recB recC genotype. Intraplasmidic recombination is recF dependent in cells having a functional exonuclease V, but not in recB recC mutants. Exonuclease V activity inhibits both interplasmidic and intraplasmidic recombination via the recE pathway.     

Specialized transduction with lambda plac5: dependence on recB.

Genetically disabled lambda plac5 transducing phage derivatives were used to study the recB dependence of recombination during specialized transduction. The frequency of transduction was normalized to colony-forming units, and the end product of recombination was monitored by scoring for addition and substitution transductants. When a chromosomal lac gene was the recipient DNA substrate molecule, both the normalized transduction frequency and the proportion of addition and substitution transductants showed essentially no recB dependence. There was a pronounced recB dependence for both normalized transduction frequency and recombination end product formation when F42 lac was the recipient DNA substrate. recB appears to have no significant role in the recombination that occurs between the two lac regions in an addition transductant. UV irradiation of the transducing phages increased the absolute level of both addition and substitution transductants obtained with a chromosomal lac gene but resulted in a considerable change in the relative frequency of addition versus substitution transductants.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.     

Suppression of Escherichia coli recF mutations by recA-linked srfA mutations.

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.