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Recombination Hotspot ANDREW F Proc. Natd. Acad. Sci. USA Vol. 89, pp. 5226-5230, June 1992 Biochemistry RecBCD enzyme is altered upon cutting DNA at a Chi recombination hotspot ANDREW F. TAYLOR AND GERALD R. SMITH Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104 Communicated by Hamilton 0. Smith, March 16, 1992 ABSTRACT During its unidirectional unwinding of DNA, to ensure even numbers of exchanges in conjugal and trans- RecBCD enzyme cuts one DNA strand near a properly oriented ductional crosses (13). At each end ofthe donor fragment one Chi site, a hotspot of homologous genetic recombination in RecBCD molecule is proposed to enter and promote just one Escherichia cohl. We report here that individual DNA molecules exchange. Such a mechanism would ensure exactly two containing two properly oriented Chi sites were cut with about exchanges and, hence, viability. 40% efficiency at one or the other Chi site but not detectably By what mechanism might RecBCD promote just one at both Chi sites. Furthermore, initial incubation of RecBCD exchange near each end ofthe donor fragment? This problem with Chi-containing DNA reduced its ability both to unwind is compounded by the high density of Chi sites in E. coli DNA and to cut at Chi sites on subsequently added DNA DNA: Chi occurs, on the average, once every 5 kilobases (kb) molecules much more than did initial incubation with Chi-free (18). A conjugational donor fragment one-quarter of the DNA; the nuclease activity was less severely affected. These chromosome long would contain about 250 Chi sites. Coor- results imply that RecBCD loses its Chi-cutting activity upon dination among the multitude of potential exchanges at these cutting at a single Chi site and provide a mechanism for sites seems difficult. Here we report that RecBCD, upon ensuring single genetic exchanges near the ends of DNA nicking DNA at a Chi site, loses the ability to nick DNA at molecules. a second Chi site. This observation provides a simple mech- anism for ensuring exactly two exchanges in conjugal and RecBCD (EC 3.1.11.5) is a multifunctional enzyme required transductional crosses. for homologous recombination by the major (RecBCD) path- way of Escherichia coli (reviewed in refs. 1 and 2). The MATERIALS AND METHODS enzyme unwinds linear duplex DNA, from a flush or nearly Growth of Plasmids and Bacteriophages. Plasmids pBR322 flush duplex end (3), with the production of single-stranded x,0 pBR322 X+E224 (19), pBR322 y+F225 (19), pBR322 X+F (ss) DNA loops that enlarge at about 100 nucleotides (nt) per X+H (ref. 12; D. Dixon, personal communication), and sec as the enzyme travels along the DNA at about 300 nt per pBR322 y+E224 y+F225 (constructed by ligation of appro- sec (4, 5). The enzyme hydrolyses about two ATP molecules priate fragments from pBR322 y+E224 and pBR322 y+F225) per base pair unwound (6). When the enzyme encounters a were grown by chloramphenicol-induced amplification in E. Chi site, 5'-GCTGGTGG-3', it frequently nicks the Chi- coli N100 (galK recA thyA) and purified by alkaline lysis (20), containing strand about 5 nt to the 3' side of Chi (7, 8). followed by banding twice in cesium chloride/ethidium bro- Nicking at Chi occurs ifthe enzyme approaches Chi from the mide equilibrium density gradients. Bacteriophage A b2 cI857 right, as the sequence is written here, but not ifit approaches X+C151 susS7 (21) was grown by lytic infection of strain Chi from the left (8). DNA unwinding is postulated to JC8679 (F- A- thr-J leu-6 thi-) lacYI galK2 ara-14 xyl-5 continue after Chi cutting (as shown in Fig. 1), with 3'-ended proA2 his4 argE3 rpsL31 tsx-33 mtl-i recB21 recC22 sbcA23 ss DNA, bearing Chi near its end, being extruded as the supE44; ref. 22). Phage were purified (21) and DNA was enzyme continues to travel along DNA (9). This ss DNA isolated by phenol extraction and dialysis. "tail" has been proposed (9) to be a potent substrate for RecBCD Enzyme. RecBCD was purified and assayed as RecA protein, which, together with SSB (ss DNA-binding described (23, 24). The specific activity was 325,000 ds protein), forms joint molecules between ss DNA and homol- exonuclease units per mg ofprotein, which was measured by ogous double-stranded (ds) DNA (reviewed in ref. 10). The A280 and the molar extinction coefficient calculated for concomitant action of purified RecBCD enzyme, RecA pro- RecBCD (5). A conversion factor of 5.6 x 109 enzyme tein, and SSB produces joint molecules from linear ds DNA molecules per ds exonuclease unit was used, calculated from and circular, supercoiled ds DNA (11). A Chi site in the linear the specific activity noted above, from the enzyme's subunit ds DNA determines the apparent size of these joint mole- molecular weights deduced from the DNA sequence (25-27), cules: thejoint molecules are apparently larger the farther the and on the assumption that the active form of the enzyme is Chi site is from one end of the linear ds DNA (the end a heterotrimer. "downstream" or 5' of the Chi site) (12). DNA Substrates. (i) Mid-labeled two-Chi substrates. Plas- The RecBCD pathway is the principal pathway of recom- mid pBR322 and its X+E and X+E X+F derivatives were bination during conjugation and transduction of E. coli. In linearized with Sty I and then 32P-labeled at the 5' termini by either case, a linear duplex chromosomal fragment from the treatment with calf intestinal phosphatase followed by incu- donor recombines with a complete circular chromosome in bation with [y_32P]ATP (New England Nuclear; 3000 Ci/ the recipient cell. Consequently, two (or any even number of) mmol; 1 Ci = 37 GBq) and polynucleotide kinase. Following exchanges are required to produce a viable (complete, cir- thermal inactivation of the polynucleotide kinase, the labeled cular) recombinant chromosome. The model in Fig. 1, based DNAs were cut with EcoRI. After thermal inactivation of the on one initially proposed for recombination of vegetative EcoRI, the DNA was mixed with a 10-fold molar excess of an phage A via the RecBCD pathway (9), provides a mechanism EcoRI "Taylomere" (23), a synthetic oligonucleotide that generates an EcoRI site on base-pairing with itself. Addition of The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: nt, nucleotide(s); ss, single-stranded; ds, double- in accordance with 18 U.S.C. §1734 solely to indicate this fact. stranded; SSB, single-stranded DNA-binding protein from E. coli. 5226 Downloaded by guest on September 30, 2021 Biochemistry: Taylor and Smith Proc. Natl. Acad. Sci. USA 89 (1992) 5227 A Loop-tail Twin-loop Chi nick I :.: - i Q - - ('hi 5 6 -:= =. - - - _ mU --~~MM Mm t m m D-loop Holliday junlctionl B __ ~ Z ....m. -C * 3mm** , - _ FIG. 1. A model of conjugal recombination promoted by RecBCD enzyme and Chi sites (13). Hfr DNA enters the recipient cell as a single strand and is rapidly converted to duplex DNA (14). (A) RecBCD-mediated interaction between one end of the linear duplex Hfr DNA (thin lines) and part of the circular F- chromosomal DNA (thick lines) is shown. (Step 1) The enzyme (stippled box) attaches to the ds DNA end and travels along the DNA, unwinding the 3'-terminthted strand and releasing it at a slower rate, to form a loop-tail structure. (Step 2) Base pairing between the extruded tails produces a twin-loop structure (4). (Step 3) The enzyme nicks one strand of a correctly oriented Chi site, generating a 3' ss DNA tail, with Chi at its end. (Step 4) Continued travel of RecBCD elongates the 3-terminated tail and releases the loop on the other strand to produce a gap. (Step 5) The ss tail, aided by RecA and SSB, invades the homologous region of the circular F- chromosome to form a D-loop. (Step 6) Nicking of the D-loop (possibly RecBCD-mediated; 15, 16), followed by strand annealing and ligation of the nicked strands, produces a Holliday junction. (B) Result of this scheme occurring at both ends of the duplex Hfr fragment: the Hfr DNA linked to the F- DNA by two Holliday junctions. Appropriate resolution of the junctions (possibly by the recG or ruvC gene products; ref. 17), as shown by the open arrowheads, produces a recombinant with the Hfr DNA substituted for part of the F- chromosome. In an alternative mode of resolution, the 3' ends of the D-loops (A, step 5) prime replication, which proceeds around the chromosome to generate a dimeric chromosome containing two Holliday junctions, resolution of which generates one recombinant and one recipient-type chromosome (see ref. 13 for details). Recombination during transduction and transformation is proposed to occur by the same mechanism. T4 DNA ligase and ATP to the mixture resulted both in Quantitation by Autoradiography. Several timed expo- religation of the (now 32P-labeled) Sty I site and in ligation of sures, using preexposed film, were analyzed with the "Whole the Taylomeres onto the EcoRI sites at the ends of the DNA. Band Analysis" feature ofthe Visage 2000 System (Millipore) Inactivation of the DNA ligase, followed by restriction with video densitometer. For all visible bands, the background- Nde I, produced two self-complementary DNA molecules, fitting algorithm was used to estimate the intensity of the one 2300 base pairs (bp) and 32P-labeled, the other 2000 bp and band above the visible lane background. The integrated unlabeled. The two DNA species were separated from each intensities for the multiple exposures of each band were other and from DNA products in which one or more of the plotted against the fraction of the radioactivity that had ligation reactions had failed by preparative electrophoresis in decayed during the exposure, and the greatest slope observed an alkaline agarose gel.
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