Role of Erf Recombinase in P22-Mediated Plasmid Transduction

In the absence of host RecA function, plasmid transduction by bacteriophage P22 can be mediated by Erf recombinase. Erf is not carried on the infecting particle but synthesized upon infection. In the recipient cell, Erf can promote both generalized plasmid transduction (which requires the circularization of plasmids transduced as linear multimers) and specialized plasmid transduction (which requires the release of plasmid DNA from linear plasmid-phage cointegrates). Both processes of Erf-mediated plasmid transduction require host RecBCD function. In contrast, RecBCD is not required for Erf-mediated circularization of P22 DNA.     

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.     

Suppression of recA deficiency in plasmid recombination by bacteriophage lambda beta protein in RecBCD- ExoI- Escherichia coli cells.

Plasmid recombination, like other homologous recombination in Escherichia coli, requires RecA protein in most conditions. We have found that the plasmid recombination defect in a recA mutant can be efficiently suppressed by the beta protein of bacteriophage lambda. beta protein is required for homologous recombination of lambda chromosomes during lytic phage growth in a recA host and is known to have a strand-annealing activity resembling that of RecA protein. The bioluminescence recombination assay was used for genetic analysis of beta-protein-mediated plasmid recombination. Efficient suppression of the recA mutation by beta protein required the absence of the E. coli nucleases exonuclease I and RecBCD nuclease. These nucleases inhibit a RecA-mediated plasmid recombination pathway that is more efficient than the pathway functioning in wild-type cells. Like RecA-mediated plasmid recombination in RecBCD- ExoI- cells, beta-protein-mediated plasmid recombination depended on concurrent DNA replication and on the activity of the recQ gene. However, unlike RecA-mediated plasmid recombination, beta-protein-mediated recombination in RecBCD- ExoI- cells was independent of recF and recJ activities. We propose that inactivation of exonuclease I and RecBCD nuclease stabilizes a recombination intermediate that is involved in RecA- and beta-protein-catalyzed homologous pairing reactions. We suggest that the intermediate may be linear plasmid DNA with a protruding 3' end, since these nucleases are known to interfere with the synthesis of such linear forms. The different recF and recJ requirements for beta-protein-dependent and RecA-dependent recombinations imply that the mechanisms of formation or processing of the putative intermediate differ in the two cases.     

Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination

Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination. Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not. In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand. In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity. These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway. This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently.     

Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism.

Two novel types of alleviation of DNA restriction by the EcoKI restriction endonuclease are described. The first type depends on the presence of the gam gene product (Gam protein) of bacteriophage lambda. The efficiency of plating of unmodified phage lambda is greatly increased when the restricting Escherichia coli K-12 host carries a gam+ plasmid. The effect is particularly striking in wild-type strains and, to a lesser extent, in the presence of sbcC and recA mutations. In all cases, Gam-dependent alleviation of restriction requires active recBCD genes of the host and recombination (red) genes of the infecting phage. The enhanced capacity of Gam-expressing cells to repair DNA strand breaks might account for this phenomenon. The second type is caused by the presence of a plasmid in a restricting host lacking RecBCD enzyme. Commonly used plasmids such as the cloning vector pACYC184 can produce such an effect in strains carrying recB single mutations or in recBC sbcBC strains. Plasmid-mediated restriction alleviation in recBC sbcBC strains is independent of the host RecF, RecJ, and RecA proteins and phage recombination functions. The presence of plasmids can also relieve restriction in recD strains. This effect depends, however, on the RecA function in the host. The molecular mechanism of the plasmid-mediated restriction alleviation remains unclear.     

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.     

The P22 Erf protein and host RecA provide alternative functions for transductional segregation of plasmid-borne duplications

A tandem DNA duplication carried on a ColE1-derived plasmid segregates at high frequency upon generalized transduction by phage P22 HT. Transductional segregation of the plasmid-borne duplication can be promoted either by RecA or by the Erf function of P22, indicating that transductional segregation is a consequence of the recombination events that re-circularize the plasmid in the recipient cell. RecA-mediated and Erf-mediated transduction give similar frequencies of duplication segregation and yield the same types of segregation products, indicating that two distinct recombination machineries (RecA + RecBCD and Erf + RecBCD) perform similar or identical recombination reactions on plasmid DNA substrates transduced by bacteriophage P22 HT. Received: 4 September 1997 / Accepted: 23 March 1998     

Role of the recJ gene product in UV-induced illegitimate recombination at the hotspot.

Illegitimate recombination between a prophage and adjacent bacterial DNA is the first step in the formation of specialized transducing phage. Such recombination is rare, but it is greatly enhanced by UV irradiation. We studied the mechanism of UV-induced illegitimate recombination by examining the effect of rec mutations on the frequency of lambda bio transducing phage and found that an Escherichia coli recJ mutation reduces it by 3- to 10-fold. In addition, the recombination hotspot, which accounts for approximately 60% of lambda bio transducing phages in wild-type bacteria, was not detected in the recJ mutant. Introduction of a RecJ overexpression plasmid into the recJ mutant recovered the recombination at the hotspot. These results indicate that the RecJ protein preferentially stimulates illegitimate recombination at the hotspot. Both the hotspot and the non- hotspot sites have short regions of homology, but only the hotspot sites contain common direct-repeat sequences. We propose a model based on the 5'-3' exonuclease activity of RecJ to explain the involvement of this protein in illegitimate recombination at the hotspot.     

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.     

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.     

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.     

NinR- and red-mediated phage-prophage marker rescue recombination in Escherichia coli: recovery of a nonhomologous immlambda DNA segment by infecting lambdaimm434 phages

We examined the requirement of lambda recombination functions for marker rescue of cryptic prophage genes within the Escherichia coli chromosome. We infected lysogenic host cells with lambdaimm434 phages and selected for recombinant immlambda phages that had exchanged the imm434 region of the infecting phage for the heterologous 2.6-kb immlambda region from the prophage. Phage-encoded activity, provided by either Red or NinR functions, was required for the substitution. Red(-) phages with DeltaNinR, internal NinR deletions of rap-ninH, or orf-ninC were 117-, 12-, and 5-fold reduced for immlambda rescue in a Rec(+) host, suggesting the participation of several NinR activities. RecA was essential for NinR-dependent immlambda rescue, but had slight influence on Red-dependent rescue. The host recombination activities RecBCD, RecJ, and RecQ participated in NinR-dependent recombination while they served to inhibit Red-mediated immlambda rescue. The opposite effects of several host functions toward NinR- and Red-dependent immlambda rescue explains why the independent pathways were not additive in a Rec(+) host and why the NinR-dependent pathway appeared dominant. We measured the influence of the host recombination functions and DnaB on the appearance of orilambda-dependent replication initiation and whether orilambda replication initiation was required for immlambda marker rescue.     

Roles of RecJ,RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli

We analyzed effects of overexpression of RecE and RecT on illegitimate recombination during prophage induction in Escherichia coli and found that frequencies of spontaneous and UV-induced illegitimate recombination are enhanced by coexpression of RecE and RecT in the wild type, but the enhanced recombination was reduced by recJ, recO, or recR mutation. The results indicated that RecET-mediated illegitimate recombination depends on the functions of RecJ, RecO, and RecR, suggesting that the RecE and RecJ exonucleases play different roles in this recombination pathway and that the RecO and RecR proteins also play important roles in the recombination. On the other hand, the frequency of the RecET-mediated illegitimate recombination was enhanced by a recQ mutation, implying that the RecQ protein plays a role in suppression of RecET-mediated illegitimate recombination. It was also found that RecET-mediated illegitimate recombination is independent of the RecA function with UV irradiation, but it is enhanced by the recA mutation without UV irradiation. Based on these results, we propose a model for the roles of RecJOR on RecET-mediated illegitimate recombination.     

Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination.

A role for the RecF, RecJ, and SbcB proteins in the RecBCD-dependent recombination pathway is suggested on the basis of the effect of null recF, recJ, and sbcB mutations in Salmonella typhimurium on a "short-homology" P22 transduction assay. The assay requires recombination within short (approximately 3-kb) sequences that flank the selected marker and lie at the ends of the transduced fragment. Since these ends are subject to exonucleolytic degradation, the assay may demand rapid recombination by requiring that the exchange be completed before the essential recombining sequences are degraded. In this assay, recF, recJ, and sbcB null mutations, tested individually, cause a small decrease in recombinant recovery but all pairwise combinations of these mutations cause a 10- to 30-fold reduction. In a recD mutant recipient, which shows increased recombination, these pairwise mutation combinations cause a 100-fold reduction in recombinant recovery. In a standard transduction assay (about 20 kb of flanking sequence), recF, recJ, and sbcB mutations have a very small effect on recombinant frequency. We suggest that these three proteins promote a rate-limiting step in the RecBC-dependent recombination process. The above results were obtained with a lysogenic recipient strain which represses expression of superinfecting phage genomes and minimizes the contribution of phage recombination functions. When a nonlysogenic recipient strain is used, coinfecting phage genomes express functions that alter the genetic requirements for recombination in the short-homology assay.     

The RecF pathway of homologous recombination can mediate the initiation of DNA damage-inducible replication of the Escherichia coli chromosome.

DNA damage-inducible DNA replication in SOS-induced Escherichia coli cells, termed inducible stable DNA replication (iSDR), has previously been shown to require either the RecBCD or the RecE pathway of homologous recombination for initiation. Here, we demonstrate that recB recC sbcC quadruple mutant cells are capable of iSDR induction and that a mutation in the recJ gene abolishes the inducibility. These results indicate that the RecF pathway of homologous recombination can also catalyze iSDR initiation.     

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.     

The genetic dependence of RecBCD-Gam mediated double strand end repair in Escherichia coli

The repair of double strand breaks after gamma-irradiation in wild-type Escherichia coli lysogenic for lambda cI857 red3 is more efficient when lambda Gam protein is present. This phenomenon, called gam dependent radioresistance, requires the interaction of RecBCD enzyme and Gam protein. We compared cell survival after gamma-irradiation in wild-type and mutant lysogens with and without induction of Gam by transient heat treatment of the cells (6 min, 42 degrees C). The main conclusions are: (1) the RecBCD-Gam pathway of recombination repair is similar but not equivalent to RecBCD, a pathway operating in recD mutants; (2) the RecBCD-Gam pathway is dependent on recJ, recQ and recN gene products and it is proposed that the RecBCD-Gam complex has ability to load RecA protein onto single strand DNA.     

Genetic evidence for the requirement of RecA loading activity in SOS induction after UV irradiation in Escherichia coli

The SOS response in Escherichia coli results in the coordinately induced expression of more than 40 genes which occurs when cells are treated with DNA-damaging agents. This response is dependent on RecA (coprotease), LexA (repressor), and the presence of single-stranded DNA (ssDNA). A prerequisite for SOS induction is the formation of a RecA-ssDNA filament. Depending on the DNA substrate, the RecA-ssDNA filament is produced by either RecBCD, RecFOR, or a hybrid recombination mechanism with specific enzyme activities, including helicase, exonuclease, and RecA loading. In this study we examined the role of RecA loading activity in SOS induction after UV irradiation. We performed a genetic analysis of SOS induction in strains with a mutation which eliminates RecA loading activity in the RecBCD enzyme (recB1080 allele). We found that RecA loading activity is essential for SOS induction. In the recB1080 mutant RecQ helicase is not important, whereas RecJ nuclease slightly decreases SOS induction after UV irradiation. In addition, we found that the recB1080 mutant exhibited constitutive expression of the SOS regulon. Surprisingly, this constitutive SOS expression was dependent on the RecJ protein but not on RecFOR, implying that there is a different mechanism of RecA loading for constitutive SOS expression.     

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.     

The RecJ DNase strongly suppresses genomic integration of short but not long foreign DNA fragments by homology-facilitated illegitimate recombination during transformation of Acinetobacter baylyi

Homology-facilitated illegitimate recombination (HFIR) promotes genomic integration of foreign DNA with a single segment homologous to the recipient genome by homologous recombination in the segment accompanied by illegitimate fusion of the heterologous sequence. During natural transformation of Acinetobacter baylyi HFIR occurs at about 0.01% of the frequency of fully homologous recombination. The role of the 5' single-strand-specific exonuclease RecJ in HFIR was investigated. Deletion of recJ increased HFIR frequency about 20-fold compared with wild type while homologous recombination was not affected. Illegitimate fusion sites were predominantly located within 360 nucleotides away from the homology whereas in wild type most fusion sites were distal (500-2500 nucleotides away). RecJ overproduction reduced the HFIR frequency to half compared with wild type, and transformants with short foreign DNA segments were diminished, leading to on average 866 foreign nucleotides integrated per event (682 in wild type, 115 in recJ). In recJ always the 3' ends of donor DNA were integrated at the homology whereas in wild type these were 3' or 5'. RecJ apparently suppresses HFIR by degrading 5' non-homologous DNA tails at the post-synaptic stage. We propose that the RecJ activity level controls the HFIR frequency during transformation and the amount of foreign DNA integrated per event.