recombination hotspot - NCBI

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ANDREW F. TAYLOR AND GERALD R. SMITH. Fred Hutchinson Cancer Research Center, ... Communicated by Hamilton 0. Smith, March 16, 1992. ABSTRACT.
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

to ensure even numbers of exchanges in conjugal and transductional crosses (13). At each end of the donor fragment one RecBCD molecule is proposed to enter and promote just one exchange. Such a mechanism would ensure exactly two exchanges and, hence, viability. By what mechanism might RecBCD promote just one exchange near each end of the donor fragment? This problem is compounded by the high density of Chi sites in E. coli DNA: Chi occurs, on the average, once every 5 kilobases (kb) (18). A conjugational donor fragment one-quarter of the chromosome long would contain about 250 Chi sites. Coordination among the multitude of potential exchanges at these sites seems difficult. Here we report that RecBCD, upon nicking DNA at a Chi site, loses the ability to nick DNA at a second Chi site. This observation provides a simple mechanism for ensuring exactly two exchanges in conjugal and transductional crosses.

During its unidirectional unwinding of DNA, ABSTRACT RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia cohl. We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites. Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected. These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.

RecBCD (EC 3.1.11.5) is a multifunctional enzyme required for homologous recombination by the major (RecBCD) pathway of Escherichia coli (reviewed in refs. 1 and 2). The enzyme unwinds linear duplex DNA, from a flush or nearly flush duplex end (3), with the production of single-stranded (ss) DNA loops that enlarge at about 100 nucleotides (nt) per sec as the enzyme travels along the DNA at about 300 nt per sec (4, 5). The enzyme hydrolyses about two ATP molecules per base pair unwound (6). When the enzyme encounters a Chi site, 5'-GCTGGTGG-3', it frequently nicks the Chicontaining strand about 5 nt to the 3' side of Chi (7, 8). Nicking at Chi occurs if the enzyme approaches Chi from the right, as the sequence is written here, but not if it approaches Chi from the left (8). DNA unwinding is postulated to continue after Chi cutting (as shown in Fig. 1), with 3'-ended ss DNA, bearing Chi near its end, being extruded as the enzyme continues to travel along DNA (9). This ss DNA "tail" has been proposed (9) to be a potent substrate for RecA protein, which, together with SSB (ss DNA-binding protein), forms joint molecules between ss DNA and homologous double-stranded (ds) DNA (reviewed in ref. 10). The concomitant action of purified RecBCD enzyme, RecA protein, and SSB produces joint molecules from linear ds DNA and circular, supercoiled ds DNA (11). A Chi site in the linear ds DNA determines the apparent size of these joint molecules: the joint molecules are apparently larger the farther the Chi site is from one end of the linear ds DNA (the end "downstream" or 5' of the Chi site) (12). The RecBCD pathway is the principal pathway of recombination during conjugation and transduction of E. coli. In either case, a linear duplex chromosomal fragment from the donor recombines with a complete circular chromosome in the recipient cell. Consequently, two (or any even number of) exchanges are required to produce a viable (complete, circular) recombinant chromosome. The model in Fig. 1, based on one initially proposed for recombination of vegetative phage A via the RecBCD pathway (9), provides a mechanism

MATERIALS AND METHODS Growth of Plasmids and Bacteriophages. Plasmids pBR322 x,0 pBR322 X+E224 (19), pBR322 y+F225 (19), pBR322 X+F X+H (ref. 12; D. Dixon, personal communication), and pBR322 y+E224 y+F225 (constructed by ligation of appropriate fragments from pBR322 y+E224 and pBR322 y+F225) were grown by chloramphenicol-induced amplification in E. coli N100 (galK recA thyA) and purified by alkaline lysis (20), followed by banding twice in cesium chloride/ethidium bromide equilibrium density gradients. Bacteriophage A b2 cI857 X+C151 susS7 (21) was grown by lytic infection of strain JC8679 (F- A- thr-J leu-6 thi-) lacYI galK2 ara-14 xyl-5 proA2 his4 argE3 rpsL31 tsx-33 mtl-i recB21 recC22 sbcA23 supE44; ref. 22). Phage were purified (21) and DNA was isolated by phenol extraction and dialysis. RecBCD Enzyme. RecBCD was purified and assayed as described (23, 24). The specific activity was 325,000 ds exonuclease units per mg of protein, which was measured by A280 and the molar extinction coefficient calculated for RecBCD (5). A conversion factor of 5.6 x 109 enzyme molecules per ds exonuclease unit was used, calculated from the specific activity noted above, from the enzyme's subunit molecular weights deduced from the DNA sequence (25-27), and on the assumption that the active form of the enzyme is a heterotrimer. DNA Substrates. (i) Mid-labeled two-Chi substrates. Plasmid pBR322 and its X+E and X+E X+F derivatives were linearized with Sty I and then 32P-labeled at the 5' termini by treatment with calf intestinal phosphatase followed by incubation with [y_32P]ATP (New England Nuclear; 3000 Ci/ mmol; 1 Ci = 37 GBq) and polynucleotide kinase. Following thermal inactivation of the polynucleotide kinase, the labeled DNAs were cut with EcoRI. After thermal inactivation of the EcoRI, the DNA was mixed with a 10-fold molar excess of an EcoRI "Taylomere" (23), a synthetic oligonucleotide that generates an EcoRI site on base-pairing with itself. Addition of

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Abbreviations: nt, nucleotide(s); ss, single-stranded; ds, doublestranded; SSB, single-stranded DNA-binding protein from E. coli.

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Proc. Natl. Acad. Sci. USA 89 (1992)

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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 religation of the (now 32P-labeled) Sty I site and in ligation of the Taylomeres onto the EcoRI sites at the ends of the DNA. Inactivation of the DNA ligase, followed by restriction with Nde I, produced two self-complementary DNA molecules, one 2300 base pairs (bp) and 32P-labeled, the other 2000 bp and unlabeled. The two DNA species were separated from each other and from DNA products in which one or more of the ligation reactions had failed by preparative electrophoresis in an alkaline agarose gel. The 32P-labeled 2300-bp fragment was located by autoradiography and recovered by extraction with Geneclean (Bio 101, La Jolla, CA). (ii) Bgl II fragment of phage A. A DNA was digested with BgI II, the cohesive ends were annealed by incubation at 370C, and the desired 651-bp fragment (nt 38104-38754) was purified in a 4% polyacrylamide gel. The fragment was labeled at its 3' ends by incubation with Sequenase (United States Biochemical), [a-32P]dCTP (New England Nuclear; 800 Ci/mmol), and unlabeled dATP, dGTP, and dTTP and was purified by phenol extraction and ethanol precipitation. (iii) Nde I digests ofpBR322. pBR322 X0 and pBR322 X+F X+H DNA were digested with Nde I and purified by phenol extraction and ethanol precipitation. Concentrations were estimated both by A260 and by fluorimetry, with T7 DNA as a standard. Relative concentrations determined by these two methods differed by