The hybrid recombinational repair pathway operates in a χ activity deficient recC1004 mutant of Escherichia coli

Homologous recombination is a crucial process for the maintenance of genome integrity. The two main recombination pathways in Escherichia coli (RecBCD and RecF) differ in the initiation of recombination. The RecBCD enzyme is the only component of the RecBCD pathway which acts in the initiation of recombination, and possesses all biochemical activities (helicase, 5′-3′ exonuclease, χ cutting and loading of the RecA protein onto single-stranded (ss) DNA) needed for the processing of double stranded (ds) DNA breaks (DSB). When the nuclease and RecA loading activities of the RecBCD enzyme are inactivated, the proteins of the RecF recombination machinery, i.e., RecJ and RecFOR substitute for the missing 5′-3′ exonuclease and RecA loading activity respectively. The above mentioned activities of the RecBCD enzyme are regulated by an octameric sequence known as the χ site (5′-GCTGGTGG-3′). One class of recC mutations, designated recC*, leads to reduced χ cutting in vitro. The recC1004 strain (a member of the recC* mutant class) is recombination proficient and resistant to UV radiation. In this paper, we studied the effects of mutations in RecF pathway genes on DNA repair (after UV and γ radiation) and on conjugational recombination in recC1004 and recC1004 recD backgrounds. We found that DNA repair after UV and γ radiation in the recC1004 and recC1004 recD backgrounds depends on recFOR and recJ gene products. We also showed that the recC1004 mutant has reduced survival after γ radiation. This phenotype is suppressed by the recD mutation which abolishes the RecBCD dependent nuclease activity. Finally, the genetic requirements for conjugational recombination differ from those for DNA repair. Conjugational recombination in recC1004 recD mutants is dependent on the recJ gene product. Our results emphasize the importance of the canonical χ recognition activity in DSB repair and the significance of interchange between the components of two recombination machineries in achieving efficient DNA repair.     

Interaction of RecBCD Enzyme with DNA at Double-Strand Breaks Produced in UV-Irradiated Escherichia coli: Requirement for DNA End Processing

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m²). The activity decrease was seen by an increase in survival of phage T4 2⁻ of about 200-fold (phage T4 2⁻ has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb⁺) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.     

A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.     

Effects of recJ, recQ, and recFOR mutations on recombination in nuclease-deficient recB recD double mutants of Escherichia coli

The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5'-->3' exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5'-->3' exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5'-->3' exonuclease is an element of the RecF recombination machinery.     

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.     

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 Requirements for High Constitutive SOS Expression in recA730 Mutants of Escherichia coli

The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5′–3′ exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5′–3′ exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo.     

Role of the RecBCD Recombination Pathway in Salmonella Virulence

Mutants of Salmonella enterica lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933-936, 1993). The virulence-related defects of RecBC(-) mutants are not suppressed by sbcB and sbcCD mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by S. enterica may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during Salmonella infection.     

Nuclease activity is essential for RecBCD recombination in Escherichia coli

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double-stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of chi recombination hot-spots. These paradoxical roles are accommodated by chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that chi converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5' --> 3' exonuclease activity is essential for recombination by RecBCD at chi recombination hot-spots and at dsDNA ends in recD mutants. In the absence of RecB-dependent nuclease function, recombination becomes entirely dependent on the 5' --> 3' single-stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).     

DNA末端构型调控RecJ核酸外切酶活性

目的 核酸酶介导的DNA双链末端切割对同源重组修复至关重要。然而,DNA末端构型对RecJ 5’-3’核酸外切酶活性的调控尚不清楚。本研究旨在探究DNA3’端和5’端构型对RecJ核酸外切酶活性的影响及其机制。方法 为探究DNA3’端构型对RecJ核酸外切酶活性的影响,使用含有Mg²⁺的体系,对具有不同3’突出末端长度（9 nt与18 nt）和3’突出末端修饰（磷酸化和硫代磷酸酯修饰）的单链DNA分别进行RecJ核酸酶活性检测。为揭示DNA 3’端构型对RecJ外切酶活性的调控机制,在Mg²⁺缺失的体系中,使RecJ与底物结合后进行凝胶迁移实验（EMSA）。为探索其他调控因子与DNA3’端构型对RecJ的协同作用,分别检测5’端磷酸化修饰和单链DNA结合蛋白（SSB）对DNA3’突出末端修饰的影响。结果 DNA3’端构型包括突出末端的长度和修饰（磷酸化和硫代磷酸酯修饰）均会抑制RecJ外切酶活性。DNA 3’端磷酸化和硫代磷酸酯修饰通过重塑RecJ-DNA的结合模式抑制RecJ外切酶活性。DNA 5’端磷酸化修饰可增强RecJ对具有不同3’端...     

RecJ exonuclease: substrates, products and interaction with SSB

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5′ single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg²⁺ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg²⁺ confirmed that substrates with 5′ tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading ~1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5′ phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination,DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

Pathways for Homologous Recombination between Chromosomal Direct Repeats in Salmonella Typhimurium

Homologous recombination pathways probably evolved primarily to accomplish chromosomal repair and the formation and resolution of duplications by sister-chromosome exchanges. Various DNA lesions initiate these events. Classical recombination assays, involving bacterial sex, focus attention on double-strand ends of DNA. Sexual exchanges, initiated at these ends, depend on the RecBCD pathway. In the absence of RecBCD function, mutation of the sbcB and sbcC genes activates the apparently cryptic RecF pathway. To provide a more general view of recombination, we describe an assay in which endogenous DNA damage initiates recombination between chromosomal direct repeats. The repeats flank markers conferring lactose utilization (Lac(+)) and ampicillin resistance (Ap(R)); recombination generates Lac(-) Ap(S) segregants. In this assay, the RecF pathway is not cryptic; it plays a major role without sbcBC mutations. Others have proposed that single-strand gaps are the natural substrate for RecF-dependent recombination. Supporting this view, recombination stimulated by a double-strand break (DSB) in a chromosomal repeat depended on RecB function, not RecF function. Without RecBCD function, sbcBC mutations modified the RecF pathway and allowed it to catalyze DSB-stimulated recombination. Sexual recombination assays overestimate the importance of RecBCD and DSBs, and underestimate the importance of the RecF pathway.     

Exonuclease VII is involved in “reckless” DNA degradation in UV-irradiated Escherichia coli

The recA mutants of Escherichia coli exhibit an abnormal DNA degradation that starts at sites of double-strand DNA breaks (DSBs), and is mediated by RecBCD exonuclease (ExoV). This “reckless” DNA degradation occurs spontaneously in exponentially growing recA cells, and is stimulated by DNA-damaging agents. We have previously found that the xonA and sbcD mutations, which inactivate exonuclease I (ExoI) and SbcCD nuclease, respectively, markedly suppress “reckless” DNA degradation in UV-irradiated recA cells. In the present work, we show that inactivation of exonuclease VII (ExoVII) by an xseA mutation contributes to attenuation of DNA degradation in UV-irradiated recA mutants. The xseA mutation itself has only a weak effect, however, it acts synergistically with the xonA or sbcD mutations in suppressing “reckless” DNA degradation. The quadruple xseA xonA sbcD recA mutants show no sign of DNA degradation during post-irradiation incubation, suggesting that ExoVII, together with ExoI and SbcCD, plays a crucial role in regulating RecBCD-catalyzed chromosome degradation. We propose that these nucleases act on DSBs to create blunt DNA ends, the preferred substrates for the RecBCD enzyme. In addition, our results show that in UV-irradiated recF recA⁺ cells, the xseA, xonA, and sbcD mutations do not affect RecBCD-mediated DNA repair, suggesting that ExoVII, ExoI and SbcCD nucleases are not essential for the initial targeting of RecBCD to DSBs. It is possible that the DNA-blunting activity provided by ExoVII, ExoI and SbcCD is required for an exchange of RecBCD molecules on dsDNA ends during ongoing “reckless” DNA degradation.     

Host RecJ is required for growth of P22 erf bacteriophage.

Growth of bacteriophage P22 erf is known to require host RecA recombination function. We show that the RecA function is necessary but not sufficient to restore the plaque-forming ability of phage P22 erf; such mutant phage also requires host RecJ function. The residual efficiency of plaquing of P22 erf in a recJ background (0.03%) is completely abolished in recJ recB hosts (< 0.001%), suggesting that the RecBCD nuclease can provide an alternative function allowing phage growth. One tentative explanation is that circularization of P22 erf DNA mostly proceeds through the RecF pathway of recombination; however, less efficient circularization via the RecBCD pathway may also occur. In a recJ background, lysates obtained upon induction of an erf prophage show reduced yield (10%), suggesting that growth of P22 erf may require host RecJ in a step(s) other than circularization of phage DNA.     

Roles of ExoI and SbcCD Nucleases in “Reckless” DNA Degradation in recA Mutants of Escherichia coli

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this “reckless” DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding.The RecA protein plays a central role in homologous recombination and recombinational DNA repair in Escherichia coli, as well as in other bacterial species. It catalyzes the key stages of the recombination process—homologous pairing and DNA strand exchange. Cells carrying null mutations in the recA gene are completely deficient for homologous recombination and are extremely sensitive to DNA-damaging agents (for a review, see references 21, 24, and 25). Populations of recA null mutants contain a large proportion (50 to 60%) of nonviable cells, reflecting the inability of these mutants to repair spontaneously occurring DNA damage (31). Also, exponentially growing recA cells display pronounced spontaneous DNA degradation that presumably starts at the sites of DNA damage and that depends on RecBCD nuclease (ExoV) activity (5, 48). This phenotype of recA cells is aggravated after DNA-damaging treatment, such as UV irradiation (48).According to the present data, the majority of RecA-catalyzed DNA transactions in E. coli start with binding of the RecA protein onto single-stranded DNA (ssDNA) substrates. This binding is mediated by the RecBCD and/or RecFOR protein, which helps RecA to overcome hindrance imposed by the SSB protein during competition for the DNA substrate. The RecBCD and RecFOR proteins begin RecA polymerization on ssDNA, giving rise to a nucleoprotein filament that is indispensable for further recombination reactions (3, 33; reviewed in reference 44).The RecBCD enzyme is crucial for initiation of recombinational processes at double-stranded DNA (dsDNA) ends (or breaks [DSBs]) in wild-type E. coli (a set of reactions known as the RecBCD pathway) (9, 43, 44). Upon recognizing a blunt or nearly blunt dsDNA end and binding to it, RecBCD acts as a combination of powerful helicase and nuclease, thus unwinding and simultaneously degrading both strands of the DNA duplex. After encountering a specific octanucleotide sequence designated Chi, the strong 3′-5′ nuclease activity of the enzyme is attenuated and a weaker 5′-3′ nuclease activity is upregulated (1). This Chi-dependent modification allows RecBCD to create a long 3′ ssDNA tail and to direct the loading of RecA protein onto it (2, 3). In vivo data suggest that this transition of RecBCD from a nuclease to a recombinase mode of action requires the presence of the RecA protein, suggesting that the two proteins might interact (27).In wild-type E. coli cells, the RecFOR protein complex works predominantly on DNA gaps, which may arise in chromosomes due to replication forks passing over the noncoding lesions (e.g., UV-induced pyrimidine dimers) or may be present in replication forks stalled at different obstacles in DNA (44). On the other hand, the RecFOR complex has an important role in recBC sbcBC(D) mutant cells, replacing the RecA-loading activity of RecBCD during recombination reactions starting from dsDNA ends. Recombination reactions mediated by RecFOR proteins are termed the RecF (or RecFOR) pathway (44).Cells mutated in the recB and/or recC gene exhibit strong deficiency in conjugational and transductional recombination, as well as in the repair of DSBs (8, 21). These defects can be rectified by extragenic sbcB and sbcC(D) suppressor mutations that inactivate two nucleases, thus enabling full efficiency of the RecF pathway on dsDNA ends (21, 44). The sbcB gene (also designated xonA) encodes exonuclease I (ExoI), the enzyme that digests ssDNA in the 3′-5′ direction (23). The sbcC and sbcD genes encode subunits of the SbcCD nuclease, which acts both as an endonuclease that cleaves hairpin structures and as an exonuclease that degrades linear dsDNA molecules (10, 11). Inactivation of either of the two subunits leads to the loss of SbcCD enzyme activity (18).The exact mechanism of activation of the RecF pathway by sbc mutations is not completely understood. A plausible explanation is that inactivation of ExoI and SbcCD nucleases is necessary to prevent the degradation of recombinogenic 3′ DNA ends created in a RecBCD-independent manner (8, 23, 38, 45, 46). It was recently shown that the sbcB15 mutant allele (encoding a protein without nucleolytic activity) (37) is a better suppressor of the RecBCD⁻ phenotype than an sbcB deletion (50), suggesting that some nonnucleolytic activity of ExoI may also contribute to the efficiency of the RecF pathway (46, 50).ExoI and SbcCD are usually viewed as enzymes with inhibitory roles in recombination due to their deleterious actions on the RecF pathway. However, some results suggest that these enzymes could also have stimulatory roles in recombination reactions proceeding on the RecBCD pathway. Genetic experiments with UV-irradiated E. coli cells indicated that ExoI and SbcCD might be involved in blunting radiation-induced DNA ends prior to RecBC(D) action (38, 45, 46). Such a role of ExoI and SbcCD seems to be particularly critical in recD recF mutants, in which the majority of DSB repair depends on the RecBC enzyme (38). It was also suggested that the blunting roles of the two nucleases may be required during conjugational recombination (16, 46).In this work, we studied the effects of sbcB (xonA) and sbcD mutations on DNA degradation occurring spontaneously in exponentially growing recA mutant cells, as well as on DNA degradation induced in recA mutants by UV irradiation. We have demonstrated that in both cases DNA degradation is strongly reduced in recA mutants that carry in addition a combination of xonA and sbcD null mutations. The results described in this paper suggest that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells.     

DNA Structures Generated during Recombination Initiated by Mismatch Repair of Uv-Irradiated Nonreplicating Phage DNA in Escherichia Coli: Requirements for Helicase, Exonucleases, and Recf and Recbcd Functions

During infection of homoimmune Escherichia coli lysogens (``repressed infections'), undamaged non-replicating λ phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities--UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease--drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggests that the exonucleases also play other roles in generation and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria in which recombination is low--UvrD(-), ExoI(-), ExoVII(-), or RecJ(-)--displays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut(+) UvrD(+) Exo(+)) generate single-stranded regions in nonreplicating UV-irradiated DNA. Both recF and recB recC mutations strikingly reduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.     

Functions of multiple exonucleases are essential for cell viability, DNA repair and homologous recombination in recD mutants of Escherichia coli

Heterotrimeric RecBCD enzyme unwinds and resects a DNA duplex containing blunt double-stranded ends and directs loading of the strand-exchange protein RecA onto the unwound 3'-ending strand, thereby initiating the majority of recombination in wild-type Escherichia coli. When the enzyme lacks its RecD subunit, the resulting RecBC enzyme, active in recD mutants, is recombination proficient although it has only helicase and RecA loading activity and is not a nuclease. However, E. coli encodes for several other exonucleases that digest double-stranded and single-stranded DNA and thus might act in consort with the RecBC enzyme to efficiently promote recombination reactions. To test this hypothesis, I inactivated multiple exonucleases (i.e., exonuclease I, exonuclease X, exonuclease VII, RecJ, and SbcCD) in recD derivatives of the wild-type and nuclease-deficient recB1067 strain and assessed the ability of the resultant mutants to maintain cell viability and to promote DNA repair and homologous recombination. A complex pattern of overlapping and sometimes competing activities of multiple exonucleases in recD mutants was thus revealed. These exonucleases were shown to be essential for cell viability, DNA repair (of UV- and gamma-induced lesions), and homologous recombination (during Hfr conjugation and P1 transduction), which are dependent on the RecBC enzyme. A model for donor DNA processing in recD transconjugants and transductants was proposed.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

Mechanisms for gene conversion and homologous recombination: The double-strand break repair model and the successive half crossing-over model

Two mechanisms for gene conversion and homologous recombination were discussed. (1) The double-strand break repair model. A double-strand break is expanded to a gap, which is then repaired by copying a homologous sequence. The gene conversion is often accompanied by crossing-over of the flanking sequences. We obtained evidence for this model in Red pathway of bacteriophage lambda and RecE pathway of E. coli. (2) The successive half crossing-over model. Half crossing-over leaves one recombinant duplex and one or two end(s) out of two parental duplexes. The resulting ends are, in turn, recombinogenic. Successive rounds of the half crossing-over mechanism explains why apparent plasmid gene conversion in RecF pathway of E. coli is not accompanied by crossing-over. This model can explain chromosomal gene conversion if we assume that the donor is first replicated. Gene conversion during mating-type switching in yeast, antigenic variation in unicellular microorganisms, and chromosomal gene conversion in mammalian somatic cells are explained by this model. Distinguishing between these two mechanisms is important in understanding recombination in yeast and mammalian cells and also in its application to gene targeting.