Role of the Escherichia coli Recombination Hotspot, χ, in RecABCD-dependent Homologous Pairing

Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as χ sites, 5′-GCTGGTGG-3′. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a χ sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of χ-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and χ-specific single-stranded DNA fragment production are observed. Also, under these conditions, χ-specific fragments derived from both the upstream and downstream regions of the DNA strand containing χ and from cleavage of the non-χ-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD−) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to χ stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, χ itself may be a preferred site for initiation of homologous pairing in this concerted process. Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as χ sites, 5′-GCTGGTGG-3′. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a χ sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of χ-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and χ-specific single-stranded DNA fragment production are observed. Also, under these conditions, χ-specific fragments derived from both the upstream and downstream regions of the DNA strand containing χ and from cleavage of the non-χ-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD−) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to χ stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, χ itself may be a preferred site for initiation of homologous pairing in this concerted process. Homologous recombination in Escherichia coli occurs primarily through the RecBCD pathway of generalized recombination (for reviews see Smith, 1988Smith G.R. Microbiol. Rev. 1988; 52: 1-28Crossref PubMed Google Scholar, Smith, 1989Smith G.R. Cell. 1989; 58: 807-809Abstract Full Text PDF PubMed Scopus (91) Google Scholar), Clark and Clark and Sandler, 1994Clark A.J. Sandler S.J. Crit. Rev. Microbiol. 1994; 20: 125-142Crossref PubMed Scopus (175) Google Scholar, and Kowalczykowski et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). Genetic analysis demonstrates that recombination occurring through this pathway is dependent on the RecA, RecBCD, and single-stranded DNA binding (SSB)1( 1The abbreviations used are: SSBsingle-stranded DNA-bindingssDNAsingle-stranded DNAdsDNAdouble-stranded DNA.) 1The abbreviations used are: SSBsingle-stranded DNA-bindingssDNAsingle-stranded DNAdsDNAdouble-stranded DNA. proteins (Clark and Clark and Margulies, 1965Clark A.J. Margulies A.D. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 451-459Crossref PubMed Scopus (462) Google Scholar; Glassberg et al., 1979Glassberg J. Meyer R.R. Kornberg A. J. Bacteriol. 1979; 140: 14-19Crossref PubMed Google Scholar; Emmerson and Howard-Flanders, 1967Emmerson P.T. Howard-Flanders P. J. Bacteriol. 1967; 93: 1729-1731Crossref PubMed Google Scholar; Glassberg et al., 1979Glassberg J. Meyer R.R. Kornberg A. J. Bacteriol. 1979; 140: 14-19Crossref PubMed Google Scholar). Mutations in the recA gene decrease levels of recombination by as much as 6 orders of magnitude (Clark and Margulies, 1965Clark A.J. Margulies A.D. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 451-459Crossref PubMed Scopus (462) Google Scholar), mutations in the recB recombination by and Howard-Flanders, 1967Emmerson P.T. Howard-Flanders P. J. Bacteriol. 1967; 93: 1729-1731Crossref PubMed Google Scholar; Howard-Flanders and P. 53: PubMed Google Scholar), and in the gene recombination by et al., 1979Glassberg J. Meyer R.R. Kornberg A. J. Bacteriol. 1979; 140: 14-19Crossref PubMed Google Scholar). In addition to these recombination in the RecBCD pathway is enhanced by recombination χ the of to in their et al., PubMed Google Scholar; et al., PubMed Google Scholar; et al., G.R. A. Cell. Full Text PDF PubMed Scopus Google Scholar; Smith, G.R. Cell. Full Text PDF PubMed Scopus Google Scholar; et al., J. PubMed Scopus Google Scholar). single-stranded single-stranded DNA double-stranded DNA. single-stranded single-stranded DNA double-stranded DNA. of RecA and SSB proteins in recombination in vitro are (for see S.C. PubMed Scopus Google Scholar, S.C. Rev. 20: PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, S.C. Rev. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, and Kowalczykowski and Kowalczykowski and 1994Kowalczykowski S.C. Eggleston A.K. Rev. 1994; PubMed Scopus Google RecA both the of DNA between single-stranded DNA and double-stranded DNA and the of homologous ability of RecA to DNA is stimulated by SSB in vitro, and mutant RecA proteins suggest that the of DNA strand is to occurring in vivo et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and Kowalczykowski et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). RecBCD is and of the and In vitro, the and and DNA (for see G.R. Genetic for Scholar, Smith, G.R. and Scholar, Kowalczykowski et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar, and Kowalczykowski et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). In addition to its RecBCD single-stranded DNA fragments to the of the χ recombination et al., G.R. Cell. Full Text PDF PubMed Scopus Google Scholar; et al., G.R. Cell. Full Text PDF PubMed Scopus Google Smith, G.R. 1994; PubMed Scopus Google Scholar). is RecBCD from the of the χ sequence for to et al., G.R. Cell. Full Text PDF PubMed Scopus Google Scholar). ability of χ to recombination in a from the that the χ site is a DNA to the not the of the RecBCD this in the and of a fragment containing the χ sequence and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar; Dixon and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google 1994Kowalczykowski S.C. 1994; PubMed Scopus Google Scholar). of is a of the of the through with χ et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). mutations in the RecBCD enzymes that with to of recombination. of mutations as of a in the gene et al., G.R. J. Bacteriol. PubMed Google Scholar). and wild type levels of the to recombination at χ et al., G.R. Cell. Full Text PDF PubMed Scopus Google Scholar). to be mutations that primarily to the gene and are of a gene et al., G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). are to wild type in that are and are in levels of and recombination and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; et al., 1988; PubMed Google Scholar; et al., 1989; PubMed Scopus Google Scholar). the recombination the not of recombination and Smith, between and wild type RecBCD is that the mutant RecBCD are of activities in vivo and in et al., G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; G.R. Genetic for Scholar), et al., J. Bacteriol. PubMed Google Scholar). the formation of joint in vitro between substrates shown to on the RecA, RecBCD, and SSB proteins in a to as the et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of joint of through unwinding of linear by RecBCD enzyme, of the by RecA and SSB and DNA strand of homologous DNA molecule by RecA and S.C. in DNA and on and Scholar). χ the reaction in vitro with a to that from in vivo and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). of χ to the of RecBCD enzyme, the to In this we on to that joint molecule formation occurs and at a pairing the formation of χ-specific fragments is and fragments derived from both the and of χ are under these conditions, homologous pairing is by the of the RecA to DNA strand Furthermore, mutant RecBCD enzymes that not χ in vivo to joint molecule formation to the as the wild type RecBCD RecBCD enzymes as and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar), and their of at and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar). of the wild type of of from that a of and a with the et al., G.R. J. Bacteriol. PubMed Google Scholar). of this to be as by the does not to the of the in not of this of of RecBCD from that a of and a with the et al., G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of this to be as by the and are in by and the is by of the of of and by A. S. and and as by and J. Full Text PDF PubMed Google and and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar, RecA a on and J. PubMed Scopus Google of at SSB from and to of at and PubMed Scopus Google Scholar). enzymes and DNA enzymes from enzymes used as by et al., J. as by the et al., G.R. A. Cell. Full Text PDF PubMed Scopus Google Scholar), et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google from and DNA by et al., J. Scholar). of in by of at with enzymes and at the by reaction with and with and at the the fragment of DNA and with by and to a by of DNA by and and with reaction of linear RecA SSB and of wild type mutant RecBCD linear of by of and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar), and the are shown in of RecBCD is in the at and with the addition of RecBCD of for of the of wild type RecBCD, and enzymes in a of RecBCD joint molecule formation a binding et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). the binding reaction conditions used with DNA as the DNA the of the reaction with to the and on of and the through a that in with of and in of are as a of the DNA a reaction and are the of at the reaction conditions DNA and by a of and the donor linear at the of the reaction at the and to of and to the for at in and at with and a are as the of the linear and joint are as containing the fragment downstream of the χ sequence a as the strand and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). conditions for the DNA unwinding and χ-specific formation of linear SSB and RecBCD linear χ containing χ at the as presence of SSB the unwinding reaction by the DNA and the of by RecBCD and S. J. Full Text PDF PubMed Google Scholar; and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar). that the of of is the reaction not at and with the addition of and of for and to for the for joint molecule that for at in are as a of the linear of wild type and mutant RecBCD enzymes by the of the of SSB upon binding to and Kowalczykowski S.C. 1989; PubMed Scopus Google under DNA unwinding reaction decrease in a and the as and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar; et al., Eggleston A.K. Kowalczykowski S.C. J. Full Text PDF PubMed Google Scholar). formation of joint dependent on the of RecA, RecBCD, and SSB proteins the and a binding et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). reaction dependent on Mg2+ concentration, as for RecA protein-promoted pairing the of χ under these conditions not in vitro reaction conditions a on the activities of RecBCD (for the of Mg2+ to concentration, the Mg2+ concentration, the of the and J. Full Text PDF PubMed Google Scholar; Eggleston and A.K. Kowalczykowski S.C. J. PubMed Scopus Google and unwinding and are to χ-specific we the of Mg2+ on the ability of RecA, RecBCD, and SSB proteins to joint molecule the that Mg2+ on joint molecule formation as the donor DNA. of χ by RecBCD occurs of the unwinding and cleavage of not fragments that from strand cleavage in the of the χ sequence cleavage in addition to the a that to strand be and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar, Dixon and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). of homologous DNA by of these in formation of joint joint molecule formation is even though unwinding and χ at and of the is to in joint molecule formation is at with of the DNA used to joint are at Mg2+ of 6 and DNA unwinding and production are not at Mg2+ and RecA is known to at Mg2+ for joint molecule formation and and Kowalczykowski S.C. PubMed Scopus Google Scholar; et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), the Mg2+ of the reaction a Mg2+ of RecA the of joint molecule formation the of to linear 2) for both RecA and SSB proteins at its not these are to for pairing et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in a of pairing the of joint molecule formation a at with on of the linear to used in the formation of this joint molecule in joint molecule formation a of at and of the and not the of joint to the of joint is not due to the of in these of the is to of the is and the of the is is not used to joint that χ formation of joint χ-specific fragments are used for at and Mg2+ two are and are with fragments from the DNA strand the χ sequence the strand containing the of in the of to be and S. the fragments to be both χ-specific and derived from the non-χ-containing cleavage of the non-χ-containing strand χ by and and Smith, for we that RecBCD DNA with the strand the strand and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). of the by χ-specific fragments derived from the downstream of the strand that the χ sequence and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar, Dixon and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar; 1994Kowalczykowski S.C. 1994; PubMed Scopus Google Scholar). the of RecBCD is with Mg2+ and A.K. Kowalczykowski S.C. J. PubMed Scopus Google and the of χ-specific fragments is by in the Mg2+ we the of Mg2+ on the both of and of χ-specific fragments and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). RecBCD with in the presence of DNA unwinding and by addition of the conditions of and the of RecBCD is with respect to that is as as the is from the of the DNA Dixon and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). of formation is in at Mg2+ the is under conditions, is with the of In as and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar), the Mg2+ is in production of enhanced of DNA with reaction conditions in the Mg2+ the and A.K. Kowalczykowski S.C. J. PubMed Scopus Google In the Mg2+ is the of with in the production of is the Mg2+ the of is of the is under conditions at Mg2+ the is the is of the is at the Mg2+ is the DNA strand is that the production of at the Mg2+ is not due to the of the ability of RecBCD to and from both of the both binding and of to that the are with at the Mg2+ and Smith, G.R. J. PubMed Scopus Google Scholar; not that the enhanced of the DNA with reaction conditions in the Mg2+ the and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar). of these is that is and the of by RecBCD with Mg2+ and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar; Eggleston and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar; 1994Kowalczykowski S.C. 1994; PubMed Scopus Google Scholar). the of in the of on χ-specific fragment unwinding to in the linear two χ the of unwinding reaction DNA with of RecBCD at Mg2+ χ is by the of the two downstream and in production of these fragments occurs at of and fragments are and of the with of the a Mg2+ formation of χ-specific is and for the and production of is Mg2+ χ-specific fragments are with from and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). are of DNA fragments are and DNA at the as with DNA Mg2+ for the of the upstream fragment is with and of to the and upstream is to that in the of χ-specific fragments derived from the of χ is by the of to the of a cleavage a fragment that is at the downstream of χ-specific fragment production be at the Mg2+ of and of for the and decrease with a decrease in in is at Mg2+ with Mg2+ for Mg2+ of and Mg2+ are the the is fragments as and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). et al., G.R. Cell. Full Text PDF PubMed Scopus Google the production of a fragment derived from the at the their reaction conditions a Mg2+ that the formation of fragments is to Mg2+ concentration, for the et al., G.R. Cell. Full Text PDF PubMed Scopus Google Scholar; et al., Dixon D.A. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Dixon and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). of χ-specific fragments is by a between the of cleavage both and χ at Mg2+ the enhanced DNA cleavage at at Mg2+ at the Mg2+ χ-specific fragments are their is by the of by the downstream of of conditions, the DNA strand at the site is the and a with χ in of is a parallel between χ in vivo and stimulation of joint molecule formation in vitro, we the ability of mutant RecBCD enzymes to joint molecule mutant proteins the et al., G.R. J. Bacteriol. PubMed Google Scholar), is in recombination wild type levels of and the RecBCD et al., G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), is of in mutant proteins to show stimulation of recombination in mutant enzymes in pairing see 1) to stimulation of joint molecule formation and of RecBCD RecBCD that both the and the of joint molecule formation by the wild type are due to the presence of χ in the linear Eggleston and A.K. Kowalczykowski S.C. J. PubMed Scopus Google the and of joint molecule formation are and with the reaction of joint molecule formation a in for the for the a in decrease in joint molecule formation at the the RecA of joint et al., J. Full Text PDF PubMed Google Scholar; and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar). mutant enzymes DNA not and of on the derived from DNA shown in used in In with both in vivo and with their to both the and the RecBCD RecA and SSB joint molecule formation between linear and molecule formation for and for in pairing by the presence of χ the and of joint molecule formation with the mutant enzymes for containing of χ on joint molecule formation by and mutant RecBCD enzymes in a of the mutant RecBCD on joint molecule binding and in a to that in the to substrates and in the presence of RecBCD with the wild type RecBCD from is shown with for the of of the mutant enzymes to stimulation by χ in the joint molecule formation that in χ this the production of χ-specific fragments and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). 6 that the is to a that χ of is RecBCD is to a of of the by wild type RecBCD production of by the is to that by the wild type the RecBCD the wild type ability of RecBCD to χ and to from the presence of of that may from of the in the may be of the the are with of and et al., G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; et al., G.R. Cell. Full Text PDF PubMed Scopus Google Scholar; et al., G.R. J. Bacteriol. PubMed Google Scholar; G.R. Genetic for Scholar). by of the joint in joint molecule formation the mutant and RecBCD is shown in to in that the linear a χ sequence type the formation of both to by and to by the fragment downstream of joint In joint are in with the at and joint are detected. In containing the RecBCD of the DNA is the joint molecule is derived from by the formation of joint by the concerted of RecA, RecBCD, and SSB proteins is stimulated by the presence of the recombination χ in both the and of joint molecule formation in the donor linear and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar; Eggleston and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar). we this by that 1) the of χ-specific joint molecule formation that on the of production that from of for 2) the of formation is dependent on Mg2+ for χ-specific fragments derived from both the and are and the in vitro behavior of two mutant RecBCD enzymes in is with the of the and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar, Dixon and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar; Eggleston and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar, Eggleston and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar; 1994Kowalczykowski S.C. 1994; PubMed Scopus Google Scholar). in the is the enhanced formation of joint to joint see binding of the fragments by RecA is for joint molecule the stimulation of joint molecule formation from in the of χ-specific fragments to that of the fragments as a of from ability of RecA to to the as is by RecBCD enzyme, from of the RecA is the for the by χ-specific χ the of RecBCD and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar; Dixon and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar), the of χ-specific fragments is that of fragments in the presence in the of does not the for by χ-specific fragments the linear in these for the formation of joint is formation of joint is on the that the of is the and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar, Howard-Flanders and P. 53: PubMed Google Scholar, and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google a for the in joint molecule formation is that the at χ is the of the strand is to χ and is as binding of RecA to the at χ formation of a at that of a sequence of may in in the of by the χ-specific in a of joint this does not the of a does not to of joint is that the χ sequence itself may with RecA with its DNA the both the of χ-specific fragments to and their as the and under DNA pairing conditions may be to their as substrates for joint molecule the that DNA sequences in RecA protein-promoted pairing and S. and be of this in the of the of reaction conditions on the pairing two from the of Mg2+ on the is that the Mg2+ for joint molecule formation is by the Mg2+ of recA protein-promoted pairing at of Mg2+ that are to joint molecule formation is χ-specific fragment production by RecBCD is that the of χ-specific fragment production with Mg2+ concentration, to a et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar; Kowalczykowski et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar), this behavior is by the of that a decrease in the Mg2+ and A.K. Kowalczykowski S.C. J. PubMed Scopus Google and by the of the between the and activities of RecBCD of RecBCD is to on the of DNA unwinding and the and activities are to be of is to Mg2+ and Kowalczykowski S.C. 1989; PubMed Scopus Google Scholar), the of cleavage is and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar). of of a is a of both and cleavage with the as the and the cleavage the at a as does at χ and D.A. Kowalczykowski S.C. Cell. Full Text PDF PubMed Scopus Google Scholar), the of at that sequence is in to the of the Furthermore, the of that is by of χ by the in a of the χ-specific of the Mg2+ the of RecBCD enzyme, in cleavage both upstream and downstream of of by the of Mg2+ to the of χ-specific this is due to a decrease in the of cleavage by RecBCD is at DNA are in the of χ the that be downstream of the of χ-specific fragments is of the of is that the of χ-specific fragments in vitro is a between the of in the of χ and the of through the DNA of the Mg2+ the χ-specific fragment derived from the DNA strand that is at the site is et al., G.R. Cell. Full Text PDF PubMed Scopus Google Scholar). both the of is and the to χ is the of cleavage in the is with a of cleavage at of the upstream χ-specific fragment under these Finally, the in the formation of a χ-specific fragment at the Mg2+ fragment a with cleavage of the non-χ-containing χ DNA strand in the of the χ the of this as and both its formation and be and S. of the is the that the mutant and RecBCD are to the presence of χ linear in with their behavior and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; et al., G.R. J. Bacteriol. PubMed Google Scholar). the mutant enzymes are to the substrates used are the wild type at joint molecule formation in the presence of χ and joint are the for the of χ-specific is for mutant of the to joint molecule formation in the presence of χ is by a of the to to its both the to the gene et al., G.R. J. Bacteriol. PubMed Google Scholar), these suggest that at of the for χ of in the the ability of this of to levels of recombination in the of χ that are of and recombination at sequences et al., G.R. J. Bacteriol. PubMed Google Scholar). for this not the linear donor DNA used this the behavior of the to a that of the enzyme, to χ to its and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar, Eggleston and A.K. Kowalczykowski S.C. J. PubMed Scopus Google Scholar). In the RecBCD to χ-specific its ability to joint with that of the wild type as in this ability is due to the of of containing the RecBCD of the with the wild type a to the of enhanced recombination in and Smith, G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; et al., 1988; PubMed Google Scholar; et al., 1989; PubMed Scopus Google Scholar), the of RecBCD to χ-specific joint molecule formation Genetic to the that mutations in a of the enzyme, the enzyme, that to the of the RecBCD et al., 1989; PubMed Scopus Google Scholar). enzyme, of the is a DNA that DNA with χ sequences and Emmerson P.T. PubMed Scopus Google Scholar; et al., S. Emmerson P.T. J. Full Text PDF PubMed Google Scholar; and D.A. PubMed Scopus Google Scholar; Kowalczykowski et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). the of the and the RecBCD enzymes et al., 1994Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). a of the are mutations a is that the RecBCD the enzymes are suggest that χ may in the stimulation of homologous pairing that the of the of RecBCD of is as with the and the of in does not the of pairing in vitro that is with wild type RecBCD and the RecBCD even of the of joint molecule formation for the of is that for joint molecule formation by the wild type the that the by χ-specific is by RecBCD to RecA the of RecA with χ-specific fragments is as under and reaction conditions containing DNA. We are to Smith, and of the for the of RecBCD We are to for of mutant RecBCD enzymes and to and for of this