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.     

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.     

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.     

Genetic control of recombination exchange frequency in Escherichia coli K-12.

The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers. The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes). The absolute value of the scale depends on specific experimental conditions. recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates. On the contrary, these genes are responsible for the Freuld. The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations. The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges. The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3.     

Phage λ Has an Analog of Escherichia Coli Reco, Recr and Recf Genes

The RecF pathway catalyzes generalized recombination in Escherichia coli that is mutant for recBC, sbcB and sbcC. This pathway operating on conjugational recombination requires the recA, recF, recJ, recN, recO, recQ, recR, ruvA, ruvB and ruvC genes. In contrast, lambda mutant for its own recombination genes, int, red alpha and red beta, requires only the recA and recJ genes to recombine efficiently in recBC sbcB sbcC cells. Deletion of an open reading frame in the ninR region of lambda results in an additional requirement for recO, recR and recF in order to recombine in recBC sbcB sbcC mutant cells. This function, designated orf for recO-, recR- and recF-like function, is largely RecF pathway specific.     

Conjugational Recombination in Escherichia Coli: Genetic Analysis of Recombinant Formation in Hfr X F(-) Crosses

The formation of recombinants during conjugation between Hfr and F(-) strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located ~500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located >300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange (s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec(+) crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to ~1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.     

Role of constitutive and inducible repair in radiation resistance of Escherichia coli

Radiation resistance of Escherichia coil cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recE, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB 1157 and highly radiation-resistant isogenic strain Gam(r)444. An optimal balance ensuring a high gamma resistance of the Gam(r)444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.     

Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12

Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.     

Frequency of recombination exchanges in Escherichia coli K-12: genetic determinants controlling this frequency

RecF, recQ, ruv, recJ and recN genes of so called RecF pathway of recombination appear to be not silent on the RecBCD pathway also. These genes are responsible for the frequency of recombination exchanges per unit length of DNA. The list: recF::Kmr greater than recQ::Tn3 greater than ruv54 greater than recJ::Tn9 demonstrated the efficiency of inhibition of recombination exchanges by these mutations. The recN262 mutation gives a feeble contrary effect. It slightly increases the frequency of recombination exchanges.     

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.     

Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli.

The fate of heteroduplex molecules containing 5-, 7-, 9-, 192-, 410-, and 514-base loops after transformation of wild-type and various mutant strains of Escherichia coli has been examined. No evidence for repair was obtained for the wild type or for strains with mutations in the following genes: mutS, recA, recBC sbcBC, recD, recF, recJ, recN, recO, recR, recBC sbcBC recF uvrA, recG ruvC, ruvB, lexA3, lexA51, uvrA, nfo xth nth, polA(Ts), or pcnB. These results rule out the involvement of the SOS system and most known recombination and repair pathways. Repair of heteroduplex molecules containing 410- and 514-base loops was observed when a 1-base deletion-insertion mismatch was present nearby. The repair of both the mismatch and the loops was directed by the state of dam methylation of the DNA chains and was dependent on the product of the mutS gene. A high efficiency of repair (95%) was found even when the mismatch and the loops were 1,448 nucleotides apart. We conclude that multibase loops in DNA can be removed only as a consequence of corepair by dam-directed mismatch repair.     

RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.

The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.     

Formation of the heterozygous tandem duplication in the process of conjugation recombination in Escherichia coli: study of the effect of mutations for the recQ, uvrD, and recJ genes]

Stable tandem duplications were shown to originate from conjugational recombination between Escherichia coli HfrH strains carrying mutations for the deo operon. The duplications deoC deoD/deoA deoB::Tn5 usually constitute approximately 5% of the Deo+ offspring. The effect of mutations for the recQ, uvrD, and recJ genes on the frequency of duplications was studied. The CM1563 strain carrying the recQ mutation was shown to give, as a recipient, 20% of duplications in the Deo+ offspring. However, this property of CM1563 seems to depend on the presence of a spontaneous mutation of unknown nature, which also increased UV sensitivity of bacteria. The recQ mutation itself increased the frequency of duplications by less than 50%. The recJ mutation did not virtually affect the frequency of duplications. The uvrD mutation possessing the recombinogenic effect was shown to increase the frequency of deo+ recombinants and simultaneously decrease the frequency of duplications. Tandem duplications are assumed to be normal intermediates of multi-stage conjugational recombination initiated by the integration of the proximal region of the Hfr chromosome into different nonhomologous regions of the recipient chromosome.     

Role for radA/sms in recombination intermediate processing in Escherichia coli

RadA/Sms is a highly conserved eubacterial protein that shares sequence similarity with both RecA strand transferase and Lon protease. We examined mutations in the radA/sms gene of Escherichia coli for effects on conjugational recombination and sensitivity to DNA-damaging agents, including UV irradiation, methyl methanesulfonate (MMS), mitomycin C, phleomycin, hydrogen peroxide, and hydroxyurea (HU). Null mutants of radA were modestly sensitive to the DNA-methylating agent MMS and to the DNA strand breakage agent phleomycin, with conjugational recombination decreased two- to threefold. We combined a radA mutation with other mutations in recombination genes, including recA, recB, recG, recJ, recQ, ruvA, and ruvC. A radA mutation was strongly synergistic with the recG Holliday junction helicase mutation, producing profound sensitivity to all DNA-damaging agents tested. Lesser synergy was noted between a mutation in radA and recJ, recQ, ruvA, ruvC, and recA for sensitivity to various genotoxins. For survival after peroxide and HU exposure, a radA mutation surprisingly suppressed the sensitivity of recA and recB mutants, suggesting that RadA may convert some forms of damage into lethal intermediates in the absence of these functions. Loss of radA enhanced the conjugational recombination deficiency conferred by mutations in Holliday junction-processing function genes, recG, ruvA, and ruvC. A radA recG ruv triple mutant had severe recombinational defects, to the low level exhibited by recA mutants. These results establish a role for RadA/Sms in recombination and recombinational repair, most likely involving the stabilization or processing of branched DNA molecules or blocked replication forks because of its genetic redundancy with RecG and RuvABC.     

The phage lambda orf gene encodes a trans-acting factor that suppresses Escherichia coli recO, recR, and recF mutations for recombination of lambda but not of E. coli.

Bacteriophage lambda can recombine in recBC sbcB sbcC mutant cells by using its own gene orf, the Escherichia coli recO, recR, and recF genes, or both. Expression of an orf-containing plasmid complements the recombination defects of orf mutant phage. However, this clone does not complement a recO mutation for conjugational recombination or recO, recR, or recF mutations for repair of UV-induced DNA damage. A plasmid clone of orf produces a protein with an apparent molecular mass of 15 kDa.     

A mutant allele gam18, participating in the RecF repair path in Escherichia coli K-12]

Plasmid pGam18 carrying one of the cloned mutant loci, responsible for enhanced radiation resistance in the strain Escherichia coli Gamr444, was shown to increase resistance to the lethal effect of gamma-rays with a dose modification factor DMF = 2. Enhanced resistance was observed in wild-type cells and in the mutant recBC sbcB, but not recFBC sbcA. This indicates the involvement of a product of the gam18 locus in the RecF pathway of recombinational repair. The protective effect of plasmid pGam18 against radiation was completely abolished by mutations in the most RecF pathway genes (recF, recJ, recR, recO, recQ, recN, and ruvB). However, three mutations in the uvrD gene, which encodes DNA helicase II and belongs to the RecF pathway, can be partially complemented by plasmid pGam18. These data suggest that the mutant allele gam18 affects the DNA helicase II activity at the presynaptic stage of the RecF pathway-mediated repair of DNA double-stranded breaks induced by gamma-irradiation.     

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.     

UvrD limits the number and intensities of RecA-green fluorescent protein structures in Escherichia coli K-12

RecA is important for recombination, DNA repair, and SOS induction. In Escherichia coli, RecBCD, RecFOR, and RecJQ prepare DNA substrates onto which RecA binds. UvrD is a 3'-to-5' helicase that participates in methyl-directed mismatch repair and nucleotide excision repair. uvrD deletion mutants are sensitive to UV irradiation, hypermutable, and hyper-rec. In vitro, UvrD can dissociate RecA from single-stranded DNA. Other experiments suggest that UvrD removes RecA from DNA where it promotes unproductive reactions. To test if UvrD limits the number and/or the size of RecA-DNA structures in vivo, an uvrD mutation was combined with recA-gfp. This recA allele allows the number of RecA structures and the amount of RecA at these structures to be assayed in living cells. uvrD mutants show a threefold increase in the number of RecA-GFP foci, and these foci are, on average, nearly twofold higher in relative intensity. The increased number of RecA-green fluorescent protein foci in the uvrD mutant is dependent on recF, recO, recR, recJ, and recQ. The increase in average relative intensity is dependent on recO and recQ. These data support an in vivo role for UvrD in removing RecA from the DNA.     

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.