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CHI SITES, Recbc ENZYME , and GENENERALIZED RECOMBINATION

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CHI SITES, Recbc ENZYME , and GENENERALIZED RECOMBINATION STADLER SYMP. Vol. 13 (1981) University of Missouri, Columbia 25 CHI SITES, RecBC ENZYME , AND GENENERALIZED RECOMBINATION GERALD R. SMITH, DENNIS W. SCHULTZ, ANDREW F. TAYLOR and KATHLEEN TRIMAN Institute of Molecular Biology University of Oregon Eugene, Oregon 97403 SUMMARY With the goal of elucidating the moleculaP basis of genetic Pecombination, ouP laboPatoPy has studied special sites that pPomote Pecombination in theiP neighboPhood and enzymes essential fop r>ecom­ bination in Escherichia coli. The genetic pPopePties of C'hi sites of E.coli and phag~ ~,detePmined by the laboPatoPy of F. Stahl, suggest that C'hi is a Pecognition sequence fop a Pecombination-pPomoting enzyme. OuP laboPatoPy's finding that C'hi is the unique nucleotide sequence 5' G-C-T-G-G-T-G-G 3' (op its complement OP both) substantiates this view. Genetic studies suggest that C'hi nny be r>ecognized by the E.coli RecBC enzyme. Based upon electPon micPogPaphic studies of Peaction intePmediates we have pPoposed a model by which RecBC enzyme unwinds and Pewinds DNA. We discuss a mechanism whePeby RecBC's hypothesized nicking of DNA at C'hi sites during DNA unwinding can pPomote rocom­ bination in a nnnneP consistent with the established genetic pPopePties of C'hi. INTRODUCTION Determining the mechanism by which two homologous chromosomes interact to produce recombinant chromosomes has remained a challenging problem to molecular biologists. St~dies of recombination-deficient mutants have permitted the definition of pathways of recombination (reviewed by CLARK 1973) and the identification of some enzymes cata­ lyzing steps in these pathways (reviewed by RADDING 1973). Additional genetic studies have revealed special sites which influence recom­ bination in their neighborhood (reyiewed by STAHL 1979a). Understanding the molecular basis of recombination requires study both of the enzymes promoting recombination and of the special sites which are presumably recognized and acted upon by one or more of the recombination-promoting enzymes. Our laboratory is studying the RecBC pathway of recombination of Escherichia coli. In this paper we review our laboratory's recent studies on RecBC enzyme (one of the enzymes of the RecBC pathway) and on Chi sites, which stimulate the RecBC pathway. In addition, we discuss a hypothetical mechanism by which RecA protein (the other identified enzyme of the RecBC pathway), RecBC enzyme, and Chi sites may promote genetic recombination. 26 SMITH, SCHULTZ, TAYLOR & TRIMAN GENETIC PROPERTIES OF CHI SITES STAHL (1979a) has extensively reviewed the genetic properties of Chi sites. Here we will briefly discuss these properties to place in context the research described below. Chi sites were first studied as mutations in bacteriophage A which enhance the growth of A Red- Garn- mutants (HENDERSON & WEIL 1975). A Red- Garn- phages grow poorly for the following reasons~ 1) E, coli's RecBC enzyme, unless inhibited by the Garn+ protein of A, prevents A rolling circle replication (UNGER & CLARK 1972; ENQUIST & SKALKA 1973). 2) Circular monomeric A DNA is not packaged with high efficiency (ENQUIST & SKALKA 1973; IKEDA & KOBAYASHI 1979), 3) In the absence of rolling circle replication, recombination between monomer circles to produce dimers is the only route to packageable DNA, In the absence of the. A Red-dependent general recombination system, significant amounts of dimers are produced only by the E. coli RecBC pathway (ZISSLER et al. 1971; ENQUIST & SKALKA 1973). This system acts inefficiently on A, resulting in low phage yields and small plaques. Mutations creating Chi arise spontaneously and are selected in a Red- Garn- phage population since Chi sites enhance Rec recombination, resulting in higher phage yields and larger plaques (HENDERSON & WEIL 1975; MCMILIN et al . 1974; LAM et -al. 1974; MALONE & CHATTORAJ 1975), Chi sites arise by mutation at four (or more) widely separated loci in A (STAHL, CRASEMANN & STAHL 1975). Enhanced frequency of recom­ bination in the interval containing Chi demonstrates that these muta­ tions affect sites rather than diffusible products. Chi sites occur in the wild-type E, coli chromosome at an average density of once per 5000 basepairs (MALONE et al. 1978). These sites, when introduced into A, manifest recombination-enhancing properties indistinguishable from those of the Chi sites arising by mutation in A• Of the several pathways of recombination potentially available to A, only the E. coli RecBC pathway is influenced by Chi (GILLEN & CLARK 1974; STAHL & STAHL 1977), Stated another way, Chi lacks detectable activity in E, coli hosts mutant in the recA, recB or recC genes coding for the two known proteins (the RecA and RecBC enzymes discussed later) of the RecBC pathway (CLARK 1973). Chi does not measurably stimulate the E, coli RecE or RecF pathways of generalized recombination or the A Red pathway of gener alized recombination or the A Int-promoted site­ specific recombination. The occurrence in wild-type E. co li of Chi sites and of the recom­ bination pathway they stimulate makes plausible the possibility that Chi plays a role in E, coli recombination. In fact, DOWER (1980) found that Chi within repressed A prophages alters the distribution of crossovers in certain Pl-mediated transductions. Unless there is a feature of A prophages which distinguishes their DNA from the rest of E, coli DNA, one can conclude that Chi can act in E, col i crosses. With the isolation of a mutation inactivating a Chi site in an E, coli gene (TRIMAN, CHATTORAJ & SMITH 1981) we can now test the action of_ Chi in E~- co-Z i crosses in the absence of ~ . - Although Chi is a site, its effect on recombination is very dif­ ferent from the prophage attachment site (att) of ~- More than 99% of Int-promoted exchanges occur within the 15~se-pair (bp) att site Chi Sites and RecBC Enzyme 27, (ENQUIST et al. 1979·; ECHOLS & GREEN 1979). In contrast, nearly all of the Chi-stimulated exchanges occur outside the Chi site. Stimulation is maximal near Chi but extends, with decreasing magnitude, more than 10,000 bp from Chi (STAHL et al. 1975). This observation suggests that something "moves" away from Chi to the point of exchange. In A stimula­ tion is greater to the left (on the conventional A map) than 'to the right of Chi (STAHL et al. 1980). The active form of Chi is "dominant" to the inactive form. In other words, Chi in one parent is nearly as active as Chi in both (LAM et al. 1974). Perhaps even more striking is the observation that Chi in one parent is active when the other parent contains a non­ homology of >10 3 bp opposite the Chi (STAHL & STAHL 1975). This result would appear to be consistent with the view noted above that something "moves" from Chi to the point of exchange, which is necessarily outside (and to the left of) the region of non-homology. The observation that Chi sites in A act to their left prompted FAULDS et al. (1979) to invert Chi-containing DNA segments of >." Instead of acting to their right, the inverted Chi sites fail'ed to act at all. This result indicates that some other site in A acts in con­ junction with Chi. YAGIL et al. (1980) found that the active orien­ tation of Chi was the same throughout a large region of the;\ chromosome and suggested that active Chi sites may have to be correctly oriented with respect to the ends of the A chromosome. In summary, Chi sites ·l) arise by mutation at 4 loci in phage A, 2) occur in the E. coli chromosome once per 5000 bp on the average, 3) stimulate the E. coli RecBC pathway of recombination, 4) act when only one parent in the cross carries Chi, 5) act when one parent carries a large heterology opposite the Chi in the other parent, 6) act over a long distance (more than 10,000 bp), 7) act principally to their left, relative to the conventional;\ map, and 8) act only if the Chi is in one orient at ion in A • NUCLEOTIDE SEQUENCE OF CHI SITES The genetic properties of Chi sites suggest that Chi is a recogni­ tion sequence for some enzyme in the RecBC pathway. To obtain biochemi­ cal support for this view, our laboratory has determined the nucleotide sequence surrounding several Chi sites. We anticipated that if Chi is a recognition sequence these sites would have similar or identical sequen­ ces and that mutations altering the activity of Chi would be located within these common sequences. We summarize here our evidence that Chi is indeed a unique nucleotide sequence. For nucleotide sequence analysis of Chi it was necessary to iden­ tify an interval of A containing Chi and small enough for convenient nucleotide sequence determination. A solution to this problem was to locate Chi between the endpoints of deletions on the physical map using restriction endonuclease cleavage analysis of the deletion DNA. Figure 1 illustrates this approach for the first Chi site sequenced, x+c within the ell gene of/.. (SPRAGUE, FAULDS & SMITH 1978). [In the nomenclature of MALONE et al. (1978) x + designates the active form of Chi sites in A and x - the inactive form in >., while chi+ and chi- designate the active and inactive forms of Chi sites in E. coli.] The x+c mutation 28 SMITH, SCHULTZ, TAYLOR & TRIMAN Taq I Hind]: t t Deletion spi-71 Deletion spi-380 FIGURE 1, Genetic and physical localization of a Chi site, x+c in phage A, Genetic crosses placed x+c between the endpoints of deletions spi-7l and spi-380, Restriction endonuclease cleavage analyses placed sites for Taq I and HindII beyond the deletion endpoints as indicated. From SPRAGUE, FAULDS & SMITH (1978).
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