A RecA mutant, RecA(730), suppresses the recombination deficiency of the RecBC(1004)D-chi* interaction in vitro and in vivo

In Escherichia coli, homologous recombination initiated at double-stranded DNA breaks requires the RecBCD enzyme, a multifunctional heterotrimeric complex that possesses processive helicase and exonuclease activities. Upon encountering the DNA regulatory sequence, chi, the enzymatic properties of RecBCD enzyme are altered. Its helicase activity is reduced, the 3'-->5'nuclease activity is attenuated, the 5'-->3' nuclease activity is up-regulated, and it manifests an ability to load RecA protein onto single-stranded DNA. The net result of these changes is the production of a highly recombinogenic structure known as the presynaptic filament. Previously, we found that the recC1004 mutation alters chi-recognition so that this mutant enzyme recognizes an altered chi sequence, chi*, which comprises seven of the original nucleotides in chi, plus four novel nucleotides. Although some consequences of this mutant enzyme-mutant chi interaction could be detected in vivo and in vitro, stimulation of recombination in vivo could not. To resolve this seemingly contradictory observation, we examined the behavior of a RecA mutant, RecA(730), that displays enhanced biochemical activity in vitro and possesses suppressor function in vivo. We show that the recombination deficiency of the RecBC(1004)D-chi* interaction can be overcome by the enhanced ability of RecA(730) to assemble on single-stranded DNA in vitro and in vivo. These data are consistent with findings showing that the loading of RecA protein by RecBCD is necessary in vivo, and they show that RecA proteins with enhanced single-stranded DNA-binding capacity can partially bypass the need for RecBCD-mediated loading.     

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).     

A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi

In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.     

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.     

Chi hotspot activity in Escherichia coli without RecBCD exonuclease activity: implications for the mechanism of recombination

The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (5'-GCTGGTGG-3'). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and lambda phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.     

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.     

Homologous pairing in vitro stimulated by the recombination hotspot, Chi.

Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5'-GCT-GGTGG-3'. We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot. Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA. The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi. This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3' end for efficient joint molecule formation. Action at Chi generates invasive ssDNA from the 5' but not the 3' side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site. These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.     

A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase

RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. Several of its activities are regulated by the DNA sequence chi (5'-GCTGGTGG-3'), which is recognized in cis by the translocating enzyme. When RecBCD enzyme encounters chi, the intensity and polarity of its nuclease activity are changed, and the enzyme gains the ability to load RecA protein onto the chi-containing, unwound single-stranded DNA. Here, we show that interaction with chi also affects translocation by RecBCD enzyme. By observing translocation of individual enzymes along single molecules of DNA, we could see RecBCD enzyme pause precisely at chi. Furthermore, and more unexpectedly, after pausing at chi, the enzyme continues translocating but at approximately one-half the initial rate. We propose that interaction with chi results in an enzyme in which one of the two motor subunits, likely the RecD motor, is uncoupled from the holoenzyme to produce the slower translocase.     

Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition

In Escherichia coli, chi (5'-GCTGGTGG-3') is a recombination hotspot recognized by the RecBCD enzyme. Recognition of chi reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by chi interaction has been "ejection" of the RecD motor from the holoenzyme at chi. To test this proposal, we visualized individual RecBCD molecules labeled via RecD with a fluorescent nanoparticle. We could directly see these labeled, single molecules of RecBCD moving at up to 1835 bp/s (approximately 0.6 microm/s). Those enzymes translocated to chi, paused, and continued at reduced velocity, without loss of RecD. We conclude that chi interaction induces a conformational change, resulting from binding of chi to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.     

Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots

Homologous recombination occurs especially frequently near special chromosomal sites called hotspots. In Escherichia coli, Chi hotspots control RecBCD enzyme, a protein machine essential for the major pathway of DNA break-repair and recombination. RecBCD generates recombinogenic single-stranded DNA ends by unwinding DNA and cutting it a few nucleotides to the 3′ side of 5′ GCTGGTGG 3′, the sequence historically equated with Chi. To test if sequence context affects Chi activity, we deep-sequenced the products of a DNA library containing 10 random base-pairs on each side of the Chi sequence and cut by purified RecBCD. We found strongly enhanced cutting at Chi with certain preferred sequences, such as A or G at nucleotides 4–7, on the 3′ flank of the Chi octamer. These sequences also strongly increased Chi hotspot activity in E. coli cells. Our combined enzymatic and genetic results redefine the Chi hotspot sequence, implicate the nuclease domain in Chi recognition, indicate that nicking of one strand at Chi is RecBCD''s biologically important reaction in living cells, and enable more precise analysis of Chi''s role in recombination and genome evolution.     

A stimulatory RNA associated with RecBCD enzyme.

RecBCD enzyme acts in the major pathway of homologous recombination of linear DNA in Escherichia coli. The enzyme unwinds DNA and is an ATP-dependent double-strand and single-strand exonuclease and a single-strand endonuclease; it acts at Chi recombination hotspots (5'-GCTGGTGG-3') to produce a recombinogenic single-stranded DNA 3'-end. We found that a small RNA with a unique sequence of approximately 24 nt was tightly bound to RecBCD enzyme and co-purified with it. When added to native enzyme this RNA, but not four others, increased DNA unwinding and Chi nicking activities of the enzyme. In seven similarly active enzyme preparations the molar ratio of RNA molecules to RecBCD enzyme molecules ranged from 0.2 to <0.008. These results suggest that, although this unique RNA is not an essential enzyme subunit, it has a biological role in stimulating RecBCD enzyme activity.     

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.     

Plasmid recombination by the RecBCD pathway of Escherichia coli.

Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M. M. Zaman and T. C. Boles, J. Bacteriol. 176:5093-5100, 1994). Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A. J. Clark and K. B. Low, p. 155-215, in K. B. Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains. Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains. Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes. The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination. Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells.     

Further Tests of a Recombination Model in Which χ Removes the Recd Subunit from the Recbcd Enzyme of Escherichia Coli

When one of two infecting lambda phage types in a replication-blocked cross is chi + and DNA packaging is divorced from the RecBCD-chi interaction, complementary chi-stimulated recombinants are recovered equally in mass lysates only if the chi + parent is in excess in the infecting parental mixture. Otherwise, the chi 0 recombinant is recovered in excess. This observation implies that, along with the chi 0 chromosome, two chi + parent chromosomes are involved in the formation of each chi + recombinant. The trimolecular nature of chi +-stimulated recombination is manifest in recombination between lambda and a plasmid. When lambda recombines with a plasmid via the RecBCD pathway, the resulting chromosome has an enhanced probability of undergoing lambda x lambda recombination in the interval into which the plasmid was incorporated. These two observations support a model in which DNA is degraded by Exo V from cos, the sequence that determines the end of packaged lambda DNA and acts as point of entry for RecBCD enzyme, to chi, the DNA sequence that stimulates the RecBCD enzyme to effect recombination. The model supposes that chi acts by ejecting the RecD subunit from the RecBCD enzyme with two consequences. (1) ExoV activity is blocked leaving a highly recombinagenic, frayed duplex end near chi, and (2) as the enzyme stripped of the RecD subunit travels beyond chi it is competent to catalyze reciprocal recombination.     

A single mutation, RecB(D1080A,) eliminates RecA protein loading but not Chi recognition by RecBCD enzyme.

Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (chi: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 --> Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether chi sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to chi sites by inactivating in a chi-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.     

A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli . Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.     

Recombinational hotspot activity of Chi-like sequences

Chi sites, consisting of the nucleotide octamer 5' G-C-T-G-G-T-G-G 3', stimulate coliphage lambda recombination mediated by the Escherichia coli RecBC recombination pathway. In a sensitive genetic assay using phage lambda crosses, three of four Chi-like sequences tested, namely 5' A-C-T-G-G-T-G-G 3', 5' G-T-T-G-G-T-G-G 3' and 5' G-C-T-A-G-T-G-G 3', had about 6%, 11% and 38% of full Chi activity, respectively. We conclude that certain Chi-like sequences manifest a spectrum of recombinational hotspot activities and may account for RecBC-mediated generalized recombination of lambda lacking Chi sites.     

The human minisatellite consensus at breakpoints of oncogene translocations.

A reexamination of human minisatellite (hypervariable) regions following the cloning and sequencing of the new minisatellite, VTR1.1, revealed that many of these structures possessed a strongly conserved copy of the chi-like octamer, GC[A/T]GG[A/T]GG. In oncogene translocations apparently created by aberrant VDJ recombinase activity, this VTR octamer was often found within a few bases of the breakpoint (p less than 10(-10)). Three bcl2 rearrangements which occurred within 2 bp of one another were located precisely adjacent to this consensus; it defined the 5' border of that oncogene's major breakpoint cluster. Several c-myc translocations also occurred within 2 bp of this sequence. While the appearance of a chi-like element in polymorphic minisatellite sequences is consistent with a role promoting either recombination or replication slippage, the existence of such elements at sites of somatic translocations suggests chi function in site-specific recombination, perhaps as a subsidiary recognition signal in immunoglobulin gene rearrangement. We discuss the implications of these observations for mechanisms by which oncogene translocations and minisatellite sequences are generated.     

Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.

Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.     

Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria

Chi sites, 5'G-C-T-G-G-T-G-G-3', enhance homologous recombination in Escherichia coli and are activated by the RecBCD enzyme. To test the ability of Chi to be activated by analogous enzymes from other bacteria, we cloned recBCD-like genes from diverse bacteria into an E. coli recBCD deletion mutant. Clones from seven species of enteric bacteria conferred to this deletion mutant recombination proficiency, Chi hotspot activity in lambda Red- Gam- vegetative crosses, and RecBCD enzyme activities, including Chi-dependent DNA strand cleavage. Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. These results imply that Chi has been conserved as a recombination-promoting signal for RecBCD-like enzymes in enteric bacteria but not in more distantly related bacteria such as Pseudomonas spp. We discuss the possibility that other, presently unknown, nucleotide sequences serve the same function as Chi in Pseudomonas spp.