Proc. Natl. Acad. Sci. USA Vol. 89, pp. 5912-5916, July 1992 Genetics
Nonconservative recombination in Escherichia coli (homologous recombination/RecF pathway/plasmid)
NORIKo K. TAKAHASHI*, KENJI YAMAMOTO*, YOSHIHIRo KITAMURA*, SI-QIN AND ICHIZO KOBAYASHI*t
Luot, HIROSHI YOSHIKURA*,
*Department of Bacteriology, Faculty of Medicine, and tDepartment of Molecular Biology, Institute of Medical Science, University of Tokyo, Shiroganedai, Tokyo 108, Japan
Communicated by Maurice S. Fox, February 18, 1992 (received for review May 13, 1991)
Homologous recombination between two duABSTRACT plex DNA molecules might result in two duplex DNA molecules (conservative) or, alternatively, it might result in only one recombinant duplex DNA molecule (nonconservative). Here we present evidence that the mode of homologous recombination is nonconservative in an Escherichia coli strain with an active RecF pathway (a recBC sbcBC mutant). We employed plasmid substrates that enable us to recover both recombination products. These plasmids carry two mutant alleles of neo gene in direct orientation, two drug-resistance marker genes, and two compatible replication origins. After their transfer to the cells followed by immediate selection for the recombination to neo+, we could recover only one recombination product. A doublestrand break at the region of homology increased this nonconservative recombination. If a nonconservative exchange should leave an end, this end may stimulate another exchange. Such "successive half crossing-over events" can explain several recombination-related phenomena in E. coli, including the origin of plasmid linear multimers and of transcribable, nonreplicated recombination products, and also in yeast and mammalian cells.
MATERIALS AND METHODS Plasmids pKEN33, pKEN34, and pKEN32 are described in Figs. 1 and 4. pNT1 is like the neo+ cml plasmid in Fig. 1B, bottom right. pNT2 is like the neo- amp plasmid in Fig. 1B, bottom left, except that it carries only the deletion at the N terminus. pNT1 and pNT2 were formed by transformation of JC8679 with pKEN33. The following isogenic E. coli strains are from A. J. Clark: JC7623 (recB21 recC22 sbcB15 sbcC201) (6, 7), JC8111 (as JC7623 but recF143), JC12190 (as JC7623 but recJ153), and JC7940 (as JC7623 but recA142) (8, 9). BIK1212, a recN262 tyrAJ6::TnJO derivative of JC7623, was made by transduction from SP256 (10), a gift from R. Lloyd. JC8679 (recB21 recC22 sbcA23), with a similar genetic background, and two recAl strains, DH1 and DH5, have been described (1, 11, 12). The other methods have been described (1, 11, 12).
RESULTS Experimental Design. We employed the experimental design in Fig. 1B to examine whether the mode of recombination in E. coli is conservative or nonconservative. This double-origin plasmid (detailed in Fig. 1C) carries two different mutant alleles of the neo gene in direct orientation. It also carries two compatible replication origins, one from ColEl and the other from pl5A. Each of these origins is linked to a selective marker [amp (bla), for ampicillinresistance, or cml (cat), for chloramphenicol-resistance]. This plasmid is introduced into bacterial cells, and recombination to neo+ is selected immediately. If this recombination is conservative-that is, gives two products-two neo genes should be recovered. These two neo genes could be either on two small separate plasmid molecules, as illustrated in Fig. 1B (if flanking sequences have been exchanged), or on one large plasmid molecule (if flanking sequences have not been exchanged). The number of transformants resistant to kanamycin and ampicillin (colonies on agar plate containing kanamycin and ampicillin) should be close to the number of transformants resistant to kanamycin only (colonies on agar plate containing kanamycin only) in either case. If recombination is nonconservative-that is, gives only one product-only one plasmid carrying one neo+ gene should be recovered, as illustrated in Fig. 1B. (If recombination is nonconservative and without flanking exchange, the circular plasmid molecule would become linear and would not replicate.) The number of transformants resistant to kanamycin and ampicillin (colonies on agar plate containing kanamycin and ampicillin) should be much smaller than the number of transformants resistant to kanamycin (colonies on agar plate containing kanamycin) in this case.
Mechanisms of homologous recombination between two duplex DNA molecules can be classified into two types. One type of mechanism generates two duplex products. The other type of mechanism generates only one duplex product. We call the former type conservative because the number of the molecules is conserved before and after the reaction. We call the latter type nonconservative because the number is not conserved. (Fig. 1A illustrates examples of the conservative and nonconservative mechanisms). We developed a plasmid system to ask whether homologous recombination is conservative or nonconservative in Escherichia coli. We analyzed transformation and recombination of plasmid molecules that were designed to enable us to recover either one or both of the recombination products. In this work we examined the RecF pathway, defined here as the recombination mechanism active in recBC sbcBC background (4). Our results indicate that recombination in the RecF pathway is nonconservative. A double-strand break at a region of homology exaggerated this nonconservative aspect. These results led us to propose the following "successive half crossing-over" model. Nonconservative recombination between two duplexes generates one recombinant duplex and DNA end(s). This end then stimulates another round of nonconservative recombination. This model can explain several features of homologous recombination in the E. coli RecF pathway, including the accumulation of linear plasmid multimers (5). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: AmpR, ampicillin-resistant; resistant; CmlR, chloramphenicol-resistant. 5912
KanR,
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Genetics: Takahashi
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FIG. 1. (A) Conservative recombination and nonconservative recombination. (Left) When two duplex molecules are produced from two parental duplex molecules, the recombination process is called "conservative." The two products might experience reciprocal exchange (crossing-over) of flanking sequences (as shown) or might not. Gene conversion might or might not have been associated with the exchange. (Right) When only one duplex molecule is produced from two parental duplex molecules, the process is called "nonconservative." The flanking markers out of a nonconservative recombination event can have either recombinant configuration (as shown here) or parental configuration. An exchange of the latter category is defined as "half crossing-over" between the two markers. (B) Strategy. A "double-origin" plasmid, pKEN33, carries two mutant neo alleles (white arrows) in a direct orientation, two more drug-resistant marker genes (cml and amp), and two compatible replication origins. Recombination between these two neo alleles generating wild-type neo gene is selected immediately after transfer to the cells. Conservative recombination would produce two plasmids, neo+ plasmid and amp+ plasmid, as illustrated here, when there is flanking exchange. Conservative recombination would produce one plasmid that carries at least one neo+ gene and one amp+ gene when there is no flanking exchange. In either case conservative recombination makes the cell kanamycin-resistant (KanR) and ampicillin-resistant (AmpR). Nonconservative recombination reconstructing one neo+ gene would produce only the neo+ plasmid (but not the amp plasmid) and make the cell KanR but not AmpR at the same time. The restriction site coordinates and their abbreviations are as follows: Xho I, 1082 (X); Bgl II, 1750 (B); Sac II, 2764 (S); Pvu I, 8375 (P). (C) pKEN33. A double-origin plasmid, 9000 base pairs (bp) long, derived from pIK43 (1) and pACYC184 (2, 3). The top neo gene (arrow) has a 283-bp-long deletion (deletion a) (box) between two Nae I sites, which removes its C terminus. The Nae I site has been inactivated by insertion of an 8-bp-long Xho I linker sequence, 5'-CCTCGAGG. This top fragment is derived from pIK43 (EcoRI/ HindIII fragment). The bottom neo gene carries a 248-bp-long deletion (deletion b) between two Nar I sites, which has removed the other (N) terminus. This fragment is from pIK43 (Sal I/EcoRV fragment). These two deletions are separated by a 506-bp-long region. The region of homology flanking neo is indicated by the thick line (see B); there are a 1079-bp-long region downstream and a 231-bp-long region upstream. The left unique (nonhomologous) part (2295 bp long), including the ampicillin-resistance gene and the ColEl replication origin (white circle), is from pBR322 (EcoRI/Pvu II fragment). The right unique (nonhomologous) part, including the chloramphenicol-resistance gene and the plSA replication origin (black circle), is from the Sal I/HindIII fragment of pACYC184 (2, 3), but a segment 1094 bp long was deleted during construction apparently by homologous recombination between sequence repeats in pACYC184 [2181-2273 and 3275-3367 (3)]. kb, Kilobase.
Recovery of Only One Recombination Product. We grew these double-origin plasmids in a recA- strain (DH1 or DH5) with minimal generation number. We then transformed these plasmid preparations into a recA- strain (DH1) to check the neo+ recombinants. We found that the ratio of KanR transformants to AmpR transformants was always 200 bp. One is a deletion between two Nae I sites, spanning the C terminus of the neo gene, and sealed with an 8-bp Xho I linker. We replaced the deletion allele with a simple insertion of the 8-bp-long Xho I linker at the Nae I site within the coding region. The resulting plasmid, pKEN34 (see Fig. 4), gave us the same biased results as those seen for the deletion (Fig. 3B): the KanR AmpR colonies (colonies on agar plate containing kanamycin and ampicillin) were far fewer than the KanR colonies (colonies on agar containing kanamycin only). We found the expected neo+ cml+ amp- recombination product in all (12/12) of the examined KanR transformants from pKEN34 (restriction enzymes used for analysis were Pvu I, Sac II, Xho I, and Bgl II). All of these KanR transformants were Amps and CmlR as expected. In the RecE pathway strain the number of the KanR AmpR doubleresistant transformants (colonies on agar plate containing kanamycin and ampicillin) was close to that of the KanR transformants (colonies on agar plate containing kanamycin only) (data not shown). Therefore the failure to recover both of the products is not specifically due to the deletion allele. Two Recombination Products Can Coexist in the Same Ceil. When we transformed the bacterial cells (JC7623) with two plasmids, pNT1 (with neo and piSA origin) and pNT2 (with amp and ColEl origin), we obtained the KanR AmpR doubleresistant transformants (colonies on agar plate containing kanamye.o and ampicillin) at a high frequency (data not shown). We analyzed these transformants and recovered these two plasmids in two clones examined. This demon-
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strates that these two plasmids can be established together within one cell under appropriate selection. Analysis of the rare double-resistant transformants also indicated that the two predicted recombination products can be present in one cell. At a very large input of the doubleorigin plasmids (pKEN33, pKEN30, and pKEN34), we were able to recover few KanR AmpR double-resistant transformants on agar plate containing kanamycin and ampicillin (e.g., Fig. 3B). We detected the two expected recombinant plasmid molecules, one neo+ amp- (like pNT1) and the other neoamp+ (like pNT2) in each of the 10 transformants from pKEN33. We do not know the origin of these rare double-resistant (KanR AmpR) transformants. Possibilities include (i) double transformation, immediate recombination to neo+ in one molecule, and secondary recombination in the other molecule or in its descendants; (ii) rare replication of one incoming molecule followed by two independent recombination events as above; (iii) rare conservative recombination after single transformation; (iv) half crossing-over to generate one circular neo+ plasmid molecule and a linearized molecule, followed by repair of the linearized plasmid using the circular neo+ plasmid, which has replicated, as template. In any case, this result demonstrates that the two possible recombination products can stay together in one cell once they are produced and appropriately selected by antibiotics. This is the condition used in our experiments. Effect of Double-Strand Breaks. Since these double-origin plasmids carry an Xho I linker insertion mutation in one of the mutant neo alleles, treatment in vitro with Xho I restriction endonuclease generates a double-strand break (in pKEN34) or a double-strand gap of 280 bp (in pKEN33 and pKEN32). This treatment before transformation increased recombination to neo+ in all of these plasmids (Fig. 4). We recovered the expected recombinant plasmid forms in the KanR clones (12/12 for pKEN34, 12/12 for pKEN33, 12/12 for pKEN32). This stimulation by double-strand breaks apparently exag-
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FIG. 3. Transformation of a RecF pathway strain with other double-origin plasmids. (A) With pKEN32 (see Fig. 4). The procedure is as described in the legend to Fig. 2, except that KanR CmlR transformants [colonies on agar containing kanamycin and chloramphenicol (15 ,ug/ml) after two-overnight incubation] instead of KanR AmpR transformants (colonies on agar containing kanamycin and ampicillin) were scored. The relative transformation efficiencies of a neo+ cmI+ plasmid, pNT1, on Kan Cml agar and on Kan agar were 0.39 and 0.41 at inputs of 15 ng and 75 ng, respectively (the recipient was JC7623). Relative transformation efficiencies {[colonies on Cml agar (two-overnight incubation)]/colonies on Amp agar} were as follows at an input of 0.1 ,ug: pKEN33 into DH5, 0.95; pKEN34 into DH5, 1.00; pKEN32 into DH5, 0.88; pKEN33 into JC7623, 0.60; pKEN34 into JC7623, 0.42; pKEN32 into JC7623, 0.33.) (B) With pKEN34 (see Fig. 4).
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FIG. 4. Effect of a double-strand break. (Left) pKEN33. The deletion in the top neo gene is sealed with an 8-bp-long Xho I linker. Cleavage at the Xho I linker leaves a 283-bp-long double-strand gap compared with the wild-type sequence. (Center) pKEN34. Cleavage with Xho I leaves a double-strand break with the linker sequences at the ends. (Right) pKEN32. Construction of pKEN32: A BamHI linker was added to the BamHI/EcoRI fragment from pIK43 (2). This fragment was inserted into pIK39 (2) cut with BamHI. Its Nhe I/EcoRV fragment and the Sal I/HindIII fragment derived from pACYC184 [but with the 1094-bp-long deletion (see Fig. 1C legend)] were ligated after filling in with Klenow fragment. Input DNA is 0.5 ,ug per transformation.
Genetics: Takahashi
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Proc. Natl. Acad. Sci. USA 89 (1992)
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gerated the nonconservative aspect of recombination as seen in Fig. 4. The double-strand breaks and gaps did not decrease the frequency of AmpR transformant colonies drastically. This result is explained by efficient nonconservative recombination generating smaller amp plasmid molecules. The AmpR clones from the cut pKEN33 carried plasmids expected from recombination to the left of the cut site (like pNT2) in all (6/6) of the clones examined with restriction enzymes Pvu I, Sac II, Bgl II, and Xho I. The cut decreased the frequency of AmpR transformants to 3.6 x 10-3 and 5.0 x 10-3 in a recA- strain (DH5) in two independent experiments. This indicates the extent of cutting and religation in our experiments. Involvement of ReeF Pathway Genes. Bacterial recombination following conjugation is decreased by mutations in recA, recF, recJ, or recN gene in this background (recBC sbcBC) (8, 9, 15). Mutations in these genes decreased recombination to neo+ (measured as KanR/AmpR) after transfer of one of the double-origin plasmids (Fig. 5). Double-strand gaps increased recombination to neo+ but the level was not as high as in the recBC sbcBC strain. These results mean that recombination is dependent on the function of these genes in the absence and presence of a double-strand break. The breaks decreased the AmpR transformants in these strains to a greater extent than in the parental recBC sbcBC strain (Fig. 5). This indicates involvement of these genes in the recombination generating a circular amp' plasmid.
DISCUSSION
5915
recombinant duplex. One or both of the parental molecules might suffer a double-strand break in the process. An alternative possibility is that recombination is initiated by an end of a "half molecule," as illustrated in Fig. 6 Right. A conservative outcome is impossible in this case, because DNA is present only on one side of the break. An end of a linear multimer form of plasmids, known to be accumulated in this strain (5, 17), might stimulate recombination in this way. We cannot exclude the latter possibility when the transferred plasmid molecules are intact; they might replicate immediately in the cells as a rolling circle to produce an end. We can, however, exclude this possibility when the plasmid molecules carry a double-strand break and are unable to replicate. We recovered both recombination products from the double-origin plasmid molecules with a double-strand break in the RecE and A Red pathway (N.K.T., K.Y., H.Y., and I.K., unpublished). Double-strand break repair by gene conversion is a major event in the RecE and A Red pathway but cannot be a major component in the RecF pathway. We failed to detect any double-strand gap repair activity in the RecF pathway (N.K.T., K.Y., H.Y., and I.K., unpublished; refs. 12, 14). Transformation of the recBC sbcBC mutant with A DNA resulted frequently in marker replacement rather than reciprocal A incorporation (18). This early result is readily explained by the nonconservative nature of recombination revealed here. Stimulation of recombination by DNA ends in recBC sbcBC mutants was reported previously in systems that do not allow recovery of both products (19, 20). Successive Half Crossing-Over Model. Nonconservative recombination might leave an end (or two ends), as illustrated in Fig. 6. Let us define this type of recombination as half crossing-over. The resulting end(s) might stimulate another round of homologous recombination. This successive half crossing-over model can explain several mysteries in the RecF pathway of homologous recombination. (21). One mystery is the origin of the plasmid linear multimers. Half crossing-over between a replicating circle and another circle might generate a rolling circle, as illustrated in Fig. 7A. This reaction can explain why formation of these linear multimers depends on function of genes involved in the RecF pathway of recombination (recA, recF, recJ, and recO) (5, 17, 22).
1st half crossing-over
2nd half crossing-over
Nonconservative Recombination. We recovered two products of recombination after transfer of our double-origin plasmids in a recBC sbcA strain. But we were not able to do so in a recBC sbcBC strain. A simple explanation of these
results is that recombination is nonconservative in the recBC sbcBC strain. Plasmid molecules are not very stable in the absence of selection in this strain (16). Our control experiments, however, indicated that this instability cannot be the reason for our failure to recover both products of recombi-
nation. If recombination is nonconservative, how can this be so? Recombination might take place between two duplex molecules, as illustrated in Fig. 6 Left, and generate only one
FIG. 6. Successive half crossing-over model. (Left) Half crossing-over between two intact duplex molecules generating one recombinant duplex and two ends. The end engages in a second event of half crossing-over with this recombinant duplex at a slightly distant site, presumably after degradation and generation of a singlestranded tail. The final products are one duplex with two close double-strand exchanges and two ends. (Right) Half crossing-over between one intact duplex and one duplex with an end. The resulting end engages in a second event of half crossing-over as Left. The final products are one duplex with two close double-strand exchanges and one end.
5916
Genetics: Takahashi et al.
Proc. Natl. Acad. Sci. USA 89 (1992) strand tail will pair with a homologous duplex helped by recA protein and recF protein. Appropriate cutting and joining of strands will accomplish nonconservative exchange as proposed for bacterial transformation (28). Generation of one recombinant DNA molecule and two ends from two DNA molecules was proposed earlier as an intermediate step in RecBCD-mediated recombination (29). This model will be compared with ours elsewhere (26). Other Organisms. The apparent double-strand break repair experiments in yeast Saccharomyces (30, 31), which were taken as evidence for the double-strand break repair model (32, 33), can be alternatively interpreted by the successive half crossing-over model (21). Gene conversion for matingtype switching, which has been explained by the doublestrand break repair model, can also be explained by this model (21). Homologous recombination in cultured mammalian cells is likely to be end-stimulated and nonconservative (34, 35).
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FIG. 7. Implications of the successive half crossing-over model. (A) Generation of plasmid linear multimers by half crossing-over. Half crossing-over between a replicating circle and another circle generates a rolling circle. Half crossing-over between an end of a multimer (N-mer) and a circle results in a longer multimer [(N+ 1)mer]. (B) ''Transcribable recombination products" in bacterial conjugation. In the recBC strain the first half crossing-over by the RecF pathway generates a linear chromosome of the recombinant genotype. This recombinant chromosome can produce recombinant protein molecules but cannot replicate to produce recombinant progeny. A second half crossing-over in sbcBC background will restore a circular chromosome.
The second mystery is the nature of transcribable recombination products. Although recB- mutations decrease formation of viable recombinant progeny following conjugation, they do not decrease recombinant-type gene products formed immediately after conjugation (23, 24). It was proposed that recombination is arrested at the stage of the transcribable recombination product in a recB- mutant. Our successive half crossing-over model provides a different explanation (Fig. 7B). We propose that half crossing-over in the RecF pathway generates the recombinant genotype, and hence the recombinant gene product, but makes the bacterial chromosome linear. Therefore this cell cannot replicate to form a recombinant colony. In agreement with this scheme, the transcribable recombination products are reduced in the recB- recF- double mutants (24). Lloyd and Thomas (25) proposed that a linearized form of the chromosome can be one form of the transcribable recombination products. Their model assumed that the RecF pathway of recombination itself is conservative but that resolution of the Holliday-type intermediate is somehow limited to one of the two possible ways. Our present results indicate that the elementary step of RecF-mediated recombination per se is nonconservative and that it is not necessary to assume biased resolution. The third mystery is the pattern of apparent gene conversion in the RecF pathway. Apparent gene conversion in a plasmid was often found to be accompanied by crossing-over of the flanking sequences in the rec+ strain and recBC sbcA strain (1, 11). It was not accompanied by flanking exchange in a recBC sbcBC strain (26). This bias can be explained by our successive half crossing-over model. Involvement of recN, recJ, recA, and recF gene products (Fig. 5) provides us with a more concrete picture of this recombination. The action of recJ
protein,
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--
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strand exonuclease (27), at an unwound double-strand end may generate a single-strand tail with the 3' end. The single-
Drs. N. Iwata and E. Arakawa helped in plasmid preparation and in computer analysis, respectively. Drs. A. J. Clark, R. Lloyd, and S. Cohen provided us with strains and plasmids. We thank Drs. M. Fox, A. J. Clark, F. W. Stahl, K. Kusano, L. Thomason, and A. Kuzminov for comments and discussion. Dr. H. Ikeda supported us in part. The work was supported by grants to H.Y. and to I.K. from the Ministry of Education and Ministry of Health. 1. Yamamoto, K., Yoshikura, H., Takahashi, N. & Kobayashi, I. (1988) Mol. Gen. Genet. 212, 393-404. 2. Chang, A. C. Y. & Cohen, S. N. (1978) J. Bacteriol. 134, 1141-1156. 3. Rose, R. E. (1988) Nucleic Acids Res. 16, 355. 4. Clark, A. J. & Low, K. B. (1988) in The Recombination of Genetic Material, ed. Low, K. B. (Academic, New York), pp. 155-215. 5. Cohen, A. & Clark, A. J. (1986) J. Bacteriol. 167, 327-335. 6. Kushner, S. R., Nagaishi, H., Templin, A. & Clark, A. J. (1971) Proc. Natl. Acad. Sci. USA 68, 824-827. 7. Lloyd, R. G. & Buckman, C. (1985) J. Bacteriol. 164, 836-844. 8. Horii, Z. & Clark, A. J. (1973) J. Mol. Biol. 80, 327-344. 9. Lovett, S. T. & Clark, A. J. (1984) J. Bacteriol. 157, 190-196. 10. Picksley, S. M., Attfield, P. V. & Lloyd, R. G. (1984) Mol. Gen. Genet. 195, 267-274. 11. Yamamoto, K., Takahashi, N., Yoshikura, H. & Kobayashi, I. (1988) Genetics 119, 759-769. 12. Kobayashi, I. & Takahashi, N. (1988) Genetics 119, 751-757. 13. Gillen, J. R. & Clark, A. J. (1974) in Mechanisms in Genetic Recombination, ed. Grell, R. F. (Plenum, New York), pp. 123-135. 14. Takahashi, N. & Kobayashi, I. (1990) Proc. Natl. Acad. Sci. USA 87, 2790-2794. 15. Lloyd, R. G., Picksley, S. M. & Prescott, C. (1983) Mol. Gen. Genet. 190, 162-167. 16. Vapnek, D., Alton, N. K., Bassett, C. L. & Kushner, S. R. (1976) Proc. Natd. Acad. Sci. USA 73, 3492-3496. 17. Kusano, K., Nakayama, K. & Nakayama, H. (1989) J. Mol. Biol. 209, 623-634. 18. Wackernagel, W. & Radding, C. (1974) in Mechanisms in Recombination, ed. Grell, R. F. (Plenum, New York), pp. 111-122. 19. Luisi-DeLuca, C., Lovett, S. T. & Kolodner, R. D. (1989) Genetics 122, 269-278. 20. Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Thomason, L. C., Stahl, F. W. & Stahl, M. M. (1989) Genome 31, 53-67. 21. Kobayashi, I. (1992) Adv. Biophys. 28, in press. 22. Silverstein, Z. & Cohen, A. (1987) J. Bacteriol. 169, 3131-3137. 23. Birge, E. A. & Low, K. B. (1974) J. Mol. Biol. 83, 447-457. 24. Lloyd, R. G., Evans, N. P. & Buckman, C. (1987) Mol. Gen. Genet. 209,
135-141. 25. Lloyd, R. G. & Thomas, A. (1984) Mol. Gen. Genet. 197, 328-336. 26. Yamamoto, K., Kusano, K., Takahashi, N. K., Yoshikura, H. & Kobayashi, I. (1992) Mol. Gen. Genet., in press. 27. Lovett, S. T. & Kolodner, R. D. (1989) Proc. Natl. Acad. Sci. USA 86,
2627-2631. 28. Fox, M. S. (1966) J. Gen. Physiol. 49 (Suppl.), 183-196. 29. Mahan, M. J. & Roth, J. R. (1989) Genetics 121, 433-443. 30. Off-Weaver, T. L., Szostak, J. W. & Rothstein, R. J. (1981) Proc. Natl. Acad. Sci. USA 79, 6354-6358. 31. Orr-Weaver, T. L. & Szostak, J. W. (1983) Proc. Natl. Acad. Sci. USA 80, 4417-4421. 32. Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. & Stahl, F. W. (1983) Cell 33, 2S-33. 33. Resnick, M. A. (1976) J. Theor. Biol. 59, 97-106. 34. Lin, F., Sperle, K. & Sternberg, N. (1984) Mol. Cell. Biol. 4, 1020-1034. 35. Chakrabarti, S. & Seidman, M. M. (1986) Mol. Cell. Biol. 6, 2520-2526.
Nonconservative recombination in Escherichia coli (homologous recombination/RecF pathway/plasmid)
NORIKo K. TAKAHASHI*, KENJI YAMAMOTO*, YOSHIHIRo KITAMURA*, SI-QIN AND ICHIZO KOBAYASHI*t
Luot, HIROSHI YOSHIKURA*,
*Department of Bacteriology, Faculty of Medicine, and tDepartment of Molecular Biology, Institute of Medical Science, University of Tokyo, Shiroganedai, Tokyo 108, Japan
Communicated by Maurice S. Fox, February 18, 1992 (received for review May 13, 1991)
Homologous recombination between two duABSTRACT plex DNA molecules might result in two duplex DNA molecules (conservative) or, alternatively, it might result in only one recombinant duplex DNA molecule (nonconservative). Here we present evidence that the mode of homologous recombination is nonconservative in an Escherichia coli strain with an active RecF pathway (a recBC sbcBC mutant). We employed plasmid substrates that enable us to recover both recombination products. These plasmids carry two mutant alleles of neo gene in direct orientation, two drug-resistance marker genes, and two compatible replication origins. After their transfer to the cells followed by immediate selection for the recombination to neo+, we could recover only one recombination product. A doublestrand break at the region of homology increased this nonconservative recombination. If a nonconservative exchange should leave an end, this end may stimulate another exchange. Such "successive half crossing-over events" can explain several recombination-related phenomena in E. coli, including the origin of plasmid linear multimers and of transcribable, nonreplicated recombination products, and also in yeast and mammalian cells.
MATERIALS AND METHODS Plasmids pKEN33, pKEN34, and pKEN32 are described in Figs. 1 and 4. pNT1 is like the neo+ cml plasmid in Fig. 1B, bottom right. pNT2 is like the neo- amp plasmid in Fig. 1B, bottom left, except that it carries only the deletion at the N terminus. pNT1 and pNT2 were formed by transformation of JC8679 with pKEN33. The following isogenic E. coli strains are from A. J. Clark: JC7623 (recB21 recC22 sbcB15 sbcC201) (6, 7), JC8111 (as JC7623 but recF143), JC12190 (as JC7623 but recJ153), and JC7940 (as JC7623 but recA142) (8, 9). BIK1212, a recN262 tyrAJ6::TnJO derivative of JC7623, was made by transduction from SP256 (10), a gift from R. Lloyd. JC8679 (recB21 recC22 sbcA23), with a similar genetic background, and two recAl strains, DH1 and DH5, have been described (1, 11, 12). The other methods have been described (1, 11, 12).
RESULTS Experimental Design. We employed the experimental design in Fig. 1B to examine whether the mode of recombination in E. coli is conservative or nonconservative. This double-origin plasmid (detailed in Fig. 1C) carries two different mutant alleles of the neo gene in direct orientation. It also carries two compatible replication origins, one from ColEl and the other from pl5A. Each of these origins is linked to a selective marker [amp (bla), for ampicillinresistance, or cml (cat), for chloramphenicol-resistance]. This plasmid is introduced into bacterial cells, and recombination to neo+ is selected immediately. If this recombination is conservative-that is, gives two products-two neo genes should be recovered. These two neo genes could be either on two small separate plasmid molecules, as illustrated in Fig. 1B (if flanking sequences have been exchanged), or on one large plasmid molecule (if flanking sequences have not been exchanged). The number of transformants resistant to kanamycin and ampicillin (colonies on agar plate containing kanamycin and ampicillin) should be close to the number of transformants resistant to kanamycin only (colonies on agar plate containing kanamycin only) in either case. If recombination is nonconservative-that is, gives only one product-only one plasmid carrying one neo+ gene should be recovered, as illustrated in Fig. 1B. (If recombination is nonconservative and without flanking exchange, the circular plasmid molecule would become linear and would not replicate.) The number of transformants resistant to kanamycin and ampicillin (colonies on agar plate containing kanamycin and ampicillin) should be much smaller than the number of transformants resistant to kanamycin (colonies on agar plate containing kanamycin) in this case.
Mechanisms of homologous recombination between two duplex DNA molecules can be classified into two types. One type of mechanism generates two duplex products. The other type of mechanism generates only one duplex product. We call the former type conservative because the number of the molecules is conserved before and after the reaction. We call the latter type nonconservative because the number is not conserved. (Fig. 1A illustrates examples of the conservative and nonconservative mechanisms). We developed a plasmid system to ask whether homologous recombination is conservative or nonconservative in Escherichia coli. We analyzed transformation and recombination of plasmid molecules that were designed to enable us to recover either one or both of the recombination products. In this work we examined the RecF pathway, defined here as the recombination mechanism active in recBC sbcBC background (4). Our results indicate that recombination in the RecF pathway is nonconservative. A double-strand break at a region of homology exaggerated this nonconservative aspect. These results led us to propose the following "successive half crossing-over" model. Nonconservative recombination between two duplexes generates one recombinant duplex and DNA end(s). This end then stimulates another round of nonconservative recombination. This model can explain several features of homologous recombination in the E. coli RecF pathway, including the accumulation of linear plasmid multimers (5). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: AmpR, ampicillin-resistant; resistant; CmlR, chloramphenicol-resistant. 5912
KanR,
kanamycin-
Genetics: Takahashi
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Proc. Natl. Acad. Sci. USA 89 (1992) neo
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FIG. 1. (A) Conservative recombination and nonconservative recombination. (Left) When two duplex molecules are produced from two parental duplex molecules, the recombination process is called "conservative." The two products might experience reciprocal exchange (crossing-over) of flanking sequences (as shown) or might not. Gene conversion might or might not have been associated with the exchange. (Right) When only one duplex molecule is produced from two parental duplex molecules, the process is called "nonconservative." The flanking markers out of a nonconservative recombination event can have either recombinant configuration (as shown here) or parental configuration. An exchange of the latter category is defined as "half crossing-over" between the two markers. (B) Strategy. A "double-origin" plasmid, pKEN33, carries two mutant neo alleles (white arrows) in a direct orientation, two more drug-resistant marker genes (cml and amp), and two compatible replication origins. Recombination between these two neo alleles generating wild-type neo gene is selected immediately after transfer to the cells. Conservative recombination would produce two plasmids, neo+ plasmid and amp+ plasmid, as illustrated here, when there is flanking exchange. Conservative recombination would produce one plasmid that carries at least one neo+ gene and one amp+ gene when there is no flanking exchange. In either case conservative recombination makes the cell kanamycin-resistant (KanR) and ampicillin-resistant (AmpR). Nonconservative recombination reconstructing one neo+ gene would produce only the neo+ plasmid (but not the amp plasmid) and make the cell KanR but not AmpR at the same time. The restriction site coordinates and their abbreviations are as follows: Xho I, 1082 (X); Bgl II, 1750 (B); Sac II, 2764 (S); Pvu I, 8375 (P). (C) pKEN33. A double-origin plasmid, 9000 base pairs (bp) long, derived from pIK43 (1) and pACYC184 (2, 3). The top neo gene (arrow) has a 283-bp-long deletion (deletion a) (box) between two Nae I sites, which removes its C terminus. The Nae I site has been inactivated by insertion of an 8-bp-long Xho I linker sequence, 5'-CCTCGAGG. This top fragment is derived from pIK43 (EcoRI/ HindIII fragment). The bottom neo gene carries a 248-bp-long deletion (deletion b) between two Nar I sites, which has removed the other (N) terminus. This fragment is from pIK43 (Sal I/EcoRV fragment). These two deletions are separated by a 506-bp-long region. The region of homology flanking neo is indicated by the thick line (see B); there are a 1079-bp-long region downstream and a 231-bp-long region upstream. The left unique (nonhomologous) part (2295 bp long), including the ampicillin-resistance gene and the ColEl replication origin (white circle), is from pBR322 (EcoRI/Pvu II fragment). The right unique (nonhomologous) part, including the chloramphenicol-resistance gene and the plSA replication origin (black circle), is from the Sal I/HindIII fragment of pACYC184 (2, 3), but a segment 1094 bp long was deleted during construction apparently by homologous recombination between sequence repeats in pACYC184 [2181-2273 and 3275-3367 (3)]. kb, Kilobase.
Recovery of Only One Recombination Product. We grew these double-origin plasmids in a recA- strain (DH1 or DH5) with minimal generation number. We then transformed these plasmid preparations into a recA- strain (DH1) to check the neo+ recombinants. We found that the ratio of KanR transformants to AmpR transformants was always 200 bp. One is a deletion between two Nae I sites, spanning the C terminus of the neo gene, and sealed with an 8-bp Xho I linker. We replaced the deletion allele with a simple insertion of the 8-bp-long Xho I linker at the Nae I site within the coding region. The resulting plasmid, pKEN34 (see Fig. 4), gave us the same biased results as those seen for the deletion (Fig. 3B): the KanR AmpR colonies (colonies on agar plate containing kanamycin and ampicillin) were far fewer than the KanR colonies (colonies on agar containing kanamycin only). We found the expected neo+ cml+ amp- recombination product in all (12/12) of the examined KanR transformants from pKEN34 (restriction enzymes used for analysis were Pvu I, Sac II, Xho I, and Bgl II). All of these KanR transformants were Amps and CmlR as expected. In the RecE pathway strain the number of the KanR AmpR doubleresistant transformants (colonies on agar plate containing kanamycin and ampicillin) was close to that of the KanR transformants (colonies on agar plate containing kanamycin only) (data not shown). Therefore the failure to recover both of the products is not specifically due to the deletion allele. Two Recombination Products Can Coexist in the Same Ceil. When we transformed the bacterial cells (JC7623) with two plasmids, pNT1 (with neo and piSA origin) and pNT2 (with amp and ColEl origin), we obtained the KanR AmpR doubleresistant transformants (colonies on agar plate containing kanamye.o and ampicillin) at a high frequency (data not shown). We analyzed these transformants and recovered these two plasmids in two clones examined. This demon-
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strates that these two plasmids can be established together within one cell under appropriate selection. Analysis of the rare double-resistant transformants also indicated that the two predicted recombination products can be present in one cell. At a very large input of the doubleorigin plasmids (pKEN33, pKEN30, and pKEN34), we were able to recover few KanR AmpR double-resistant transformants on agar plate containing kanamycin and ampicillin (e.g., Fig. 3B). We detected the two expected recombinant plasmid molecules, one neo+ amp- (like pNT1) and the other neoamp+ (like pNT2) in each of the 10 transformants from pKEN33. We do not know the origin of these rare double-resistant (KanR AmpR) transformants. Possibilities include (i) double transformation, immediate recombination to neo+ in one molecule, and secondary recombination in the other molecule or in its descendants; (ii) rare replication of one incoming molecule followed by two independent recombination events as above; (iii) rare conservative recombination after single transformation; (iv) half crossing-over to generate one circular neo+ plasmid molecule and a linearized molecule, followed by repair of the linearized plasmid using the circular neo+ plasmid, which has replicated, as template. In any case, this result demonstrates that the two possible recombination products can stay together in one cell once they are produced and appropriately selected by antibiotics. This is the condition used in our experiments. Effect of Double-Strand Breaks. Since these double-origin plasmids carry an Xho I linker insertion mutation in one of the mutant neo alleles, treatment in vitro with Xho I restriction endonuclease generates a double-strand break (in pKEN34) or a double-strand gap of 280 bp (in pKEN33 and pKEN32). This treatment before transformation increased recombination to neo+ in all of these plasmids (Fig. 4). We recovered the expected recombinant plasmid forms in the KanR clones (12/12 for pKEN34, 12/12 for pKEN33, 12/12 for pKEN32). This stimulation by double-strand breaks apparently exag-
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FIG. 3. Transformation of a RecF pathway strain with other double-origin plasmids. (A) With pKEN32 (see Fig. 4). The procedure is as described in the legend to Fig. 2, except that KanR CmlR transformants [colonies on agar containing kanamycin and chloramphenicol (15 ,ug/ml) after two-overnight incubation] instead of KanR AmpR transformants (colonies on agar containing kanamycin and ampicillin) were scored. The relative transformation efficiencies of a neo+ cmI+ plasmid, pNT1, on Kan Cml agar and on Kan agar were 0.39 and 0.41 at inputs of 15 ng and 75 ng, respectively (the recipient was JC7623). Relative transformation efficiencies {[colonies on Cml agar (two-overnight incubation)]/colonies on Amp agar} were as follows at an input of 0.1 ,ug: pKEN33 into DH5, 0.95; pKEN34 into DH5, 1.00; pKEN32 into DH5, 0.88; pKEN33 into JC7623, 0.60; pKEN34 into JC7623, 0.42; pKEN32 into JC7623, 0.33.) (B) With pKEN34 (see Fig. 4).
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FIG. 4. Effect of a double-strand break. (Left) pKEN33. The deletion in the top neo gene is sealed with an 8-bp-long Xho I linker. Cleavage at the Xho I linker leaves a 283-bp-long double-strand gap compared with the wild-type sequence. (Center) pKEN34. Cleavage with Xho I leaves a double-strand break with the linker sequences at the ends. (Right) pKEN32. Construction of pKEN32: A BamHI linker was added to the BamHI/EcoRI fragment from pIK43 (2). This fragment was inserted into pIK39 (2) cut with BamHI. Its Nhe I/EcoRV fragment and the Sal I/HindIII fragment derived from pACYC184 [but with the 1094-bp-long deletion (see Fig. 1C legend)] were ligated after filling in with Klenow fragment. Input DNA is 0.5 ,ug per transformation.
Genetics: Takahashi
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Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 5. Involvement of rec gene functions. pKEN33, either intact (triangles) or cut with Xho I (circles), was transferred to these mutant bacterial strains by the Rb method. The input DNA was 1 ,.g, 0.7 Mg, 1 Ag, 3 ,ug, and 3 Mg, respectively. The relative transformation efficiency [(AmpR, Xho I)/(AmpR, no cut)] was as follows in our experiments: recBC sbcBC, 0.98, 0.53, 0.27; recBC sbcBC recA, 0.007, 0.024, 0.009; recBC sbcBC recF, 0.012, 0.022; recBC sbcBC recJ, 0.012, 0.007, 0.007; recBC sbcBC recN, 0.007.
gerated the nonconservative aspect of recombination as seen in Fig. 4. The double-strand breaks and gaps did not decrease the frequency of AmpR transformant colonies drastically. This result is explained by efficient nonconservative recombination generating smaller amp plasmid molecules. The AmpR clones from the cut pKEN33 carried plasmids expected from recombination to the left of the cut site (like pNT2) in all (6/6) of the clones examined with restriction enzymes Pvu I, Sac II, Bgl II, and Xho I. The cut decreased the frequency of AmpR transformants to 3.6 x 10-3 and 5.0 x 10-3 in a recA- strain (DH5) in two independent experiments. This indicates the extent of cutting and religation in our experiments. Involvement of ReeF Pathway Genes. Bacterial recombination following conjugation is decreased by mutations in recA, recF, recJ, or recN gene in this background (recBC sbcBC) (8, 9, 15). Mutations in these genes decreased recombination to neo+ (measured as KanR/AmpR) after transfer of one of the double-origin plasmids (Fig. 5). Double-strand gaps increased recombination to neo+ but the level was not as high as in the recBC sbcBC strain. These results mean that recombination is dependent on the function of these genes in the absence and presence of a double-strand break. The breaks decreased the AmpR transformants in these strains to a greater extent than in the parental recBC sbcBC strain (Fig. 5). This indicates involvement of these genes in the recombination generating a circular amp' plasmid.
DISCUSSION
5915
recombinant duplex. One or both of the parental molecules might suffer a double-strand break in the process. An alternative possibility is that recombination is initiated by an end of a "half molecule," as illustrated in Fig. 6 Right. A conservative outcome is impossible in this case, because DNA is present only on one side of the break. An end of a linear multimer form of plasmids, known to be accumulated in this strain (5, 17), might stimulate recombination in this way. We cannot exclude the latter possibility when the transferred plasmid molecules are intact; they might replicate immediately in the cells as a rolling circle to produce an end. We can, however, exclude this possibility when the plasmid molecules carry a double-strand break and are unable to replicate. We recovered both recombination products from the double-origin plasmid molecules with a double-strand break in the RecE and A Red pathway (N.K.T., K.Y., H.Y., and I.K., unpublished). Double-strand break repair by gene conversion is a major event in the RecE and A Red pathway but cannot be a major component in the RecF pathway. We failed to detect any double-strand gap repair activity in the RecF pathway (N.K.T., K.Y., H.Y., and I.K., unpublished; refs. 12, 14). Transformation of the recBC sbcBC mutant with A DNA resulted frequently in marker replacement rather than reciprocal A incorporation (18). This early result is readily explained by the nonconservative nature of recombination revealed here. Stimulation of recombination by DNA ends in recBC sbcBC mutants was reported previously in systems that do not allow recovery of both products (19, 20). Successive Half Crossing-Over Model. Nonconservative recombination might leave an end (or two ends), as illustrated in Fig. 6. Let us define this type of recombination as half crossing-over. The resulting end(s) might stimulate another round of homologous recombination. This successive half crossing-over model can explain several mysteries in the RecF pathway of homologous recombination. (21). One mystery is the origin of the plasmid linear multimers. Half crossing-over between a replicating circle and another circle might generate a rolling circle, as illustrated in Fig. 7A. This reaction can explain why formation of these linear multimers depends on function of genes involved in the RecF pathway of recombination (recA, recF, recJ, and recO) (5, 17, 22).
1st half crossing-over
2nd half crossing-over
Nonconservative Recombination. We recovered two products of recombination after transfer of our double-origin plasmids in a recBC sbcA strain. But we were not able to do so in a recBC sbcBC strain. A simple explanation of these
results is that recombination is nonconservative in the recBC sbcBC strain. Plasmid molecules are not very stable in the absence of selection in this strain (16). Our control experiments, however, indicated that this instability cannot be the reason for our failure to recover both products of recombi-
nation. If recombination is nonconservative, how can this be so? Recombination might take place between two duplex molecules, as illustrated in Fig. 6 Left, and generate only one
FIG. 6. Successive half crossing-over model. (Left) Half crossing-over between two intact duplex molecules generating one recombinant duplex and two ends. The end engages in a second event of half crossing-over with this recombinant duplex at a slightly distant site, presumably after degradation and generation of a singlestranded tail. The final products are one duplex with two close double-strand exchanges and two ends. (Right) Half crossing-over between one intact duplex and one duplex with an end. The resulting end engages in a second event of half crossing-over as Left. The final products are one duplex with two close double-strand exchanges and one end.
5916
Genetics: Takahashi et al.
Proc. Natl. Acad. Sci. USA 89 (1992) strand tail will pair with a homologous duplex helped by recA protein and recF protein. Appropriate cutting and joining of strands will accomplish nonconservative exchange as proposed for bacterial transformation (28). Generation of one recombinant DNA molecule and two ends from two DNA molecules was proposed earlier as an intermediate step in RecBCD-mediated recombination (29). This model will be compared with ours elsewhere (26). Other Organisms. The apparent double-strand break repair experiments in yeast Saccharomyces (30, 31), which were taken as evidence for the double-strand break repair model (32, 33), can be alternatively interpreted by the successive half crossing-over model (21). Gene conversion for matingtype switching, which has been explained by the doublestrand break repair model, can also be explained by this model (21). Homologous recombination in cultured mammalian cells is likely to be end-stimulated and nonconservative (34, 35).
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FIG. 7. Implications of the successive half crossing-over model. (A) Generation of plasmid linear multimers by half crossing-over. Half crossing-over between a replicating circle and another circle generates a rolling circle. Half crossing-over between an end of a multimer (N-mer) and a circle results in a longer multimer [(N+ 1)mer]. (B) ''Transcribable recombination products" in bacterial conjugation. In the recBC strain the first half crossing-over by the RecF pathway generates a linear chromosome of the recombinant genotype. This recombinant chromosome can produce recombinant protein molecules but cannot replicate to produce recombinant progeny. A second half crossing-over in sbcBC background will restore a circular chromosome.
The second mystery is the nature of transcribable recombination products. Although recB- mutations decrease formation of viable recombinant progeny following conjugation, they do not decrease recombinant-type gene products formed immediately after conjugation (23, 24). It was proposed that recombination is arrested at the stage of the transcribable recombination product in a recB- mutant. Our successive half crossing-over model provides a different explanation (Fig. 7B). We propose that half crossing-over in the RecF pathway generates the recombinant genotype, and hence the recombinant gene product, but makes the bacterial chromosome linear. Therefore this cell cannot replicate to form a recombinant colony. In agreement with this scheme, the transcribable recombination products are reduced in the recB- recF- double mutants (24). Lloyd and Thomas (25) proposed that a linearized form of the chromosome can be one form of the transcribable recombination products. Their model assumed that the RecF pathway of recombination itself is conservative but that resolution of the Holliday-type intermediate is somehow limited to one of the two possible ways. Our present results indicate that the elementary step of RecF-mediated recombination per se is nonconservative and that it is not necessary to assume biased resolution. The third mystery is the pattern of apparent gene conversion in the RecF pathway. Apparent gene conversion in a plasmid was often found to be accompanied by crossing-over of the flanking sequences in the rec+ strain and recBC sbcA strain (1, 11). It was not accompanied by flanking exchange in a recBC sbcBC strain (26). This bias can be explained by our successive half crossing-over model. Involvement of recN, recJ, recA, and recF gene products (Fig. 5) provides us with a more concrete picture of this recombination. The action of recJ
protein,
a
5'
--
3'
single-
strand exonuclease (27), at an unwound double-strand end may generate a single-strand tail with the 3' end. The single-
Drs. N. Iwata and E. Arakawa helped in plasmid preparation and in computer analysis, respectively. Drs. A. J. Clark, R. Lloyd, and S. Cohen provided us with strains and plasmids. We thank Drs. M. Fox, A. J. Clark, F. W. Stahl, K. Kusano, L. Thomason, and A. Kuzminov for comments and discussion. Dr. H. Ikeda supported us in part. The work was supported by grants to H.Y. and to I.K. from the Ministry of Education and Ministry of Health. 1. Yamamoto, K., Yoshikura, H., Takahashi, N. & Kobayashi, I. (1988) Mol. Gen. Genet. 212, 393-404. 2. Chang, A. C. Y. & Cohen, S. N. (1978) J. Bacteriol. 134, 1141-1156. 3. Rose, R. E. (1988) Nucleic Acids Res. 16, 355. 4. Clark, A. J. & Low, K. B. (1988) in The Recombination of Genetic Material, ed. Low, K. B. (Academic, New York), pp. 155-215. 5. Cohen, A. & Clark, A. J. (1986) J. Bacteriol. 167, 327-335. 6. Kushner, S. R., Nagaishi, H., Templin, A. & Clark, A. J. (1971) Proc. Natl. Acad. Sci. USA 68, 824-827. 7. Lloyd, R. G. & Buckman, C. (1985) J. Bacteriol. 164, 836-844. 8. Horii, Z. & Clark, A. J. (1973) J. Mol. Biol. 80, 327-344. 9. Lovett, S. T. & Clark, A. J. (1984) J. Bacteriol. 157, 190-196. 10. Picksley, S. M., Attfield, P. V. & Lloyd, R. G. (1984) Mol. Gen. Genet. 195, 267-274. 11. Yamamoto, K., Takahashi, N., Yoshikura, H. & Kobayashi, I. (1988) Genetics 119, 759-769. 12. Kobayashi, I. & Takahashi, N. (1988) Genetics 119, 751-757. 13. Gillen, J. R. & Clark, A. J. (1974) in Mechanisms in Genetic Recombination, ed. Grell, R. F. (Plenum, New York), pp. 123-135. 14. Takahashi, N. & Kobayashi, I. (1990) Proc. Natl. Acad. Sci. USA 87, 2790-2794. 15. Lloyd, R. G., Picksley, S. M. & Prescott, C. (1983) Mol. Gen. Genet. 190, 162-167. 16. Vapnek, D., Alton, N. K., Bassett, C. L. & Kushner, S. R. (1976) Proc. Natd. Acad. Sci. USA 73, 3492-3496. 17. Kusano, K., Nakayama, K. & Nakayama, H. (1989) J. Mol. Biol. 209, 623-634. 18. Wackernagel, W. & Radding, C. (1974) in Mechanisms in Recombination, ed. Grell, R. F. (Plenum, New York), pp. 111-122. 19. Luisi-DeLuca, C., Lovett, S. T. & Kolodner, R. D. (1989) Genetics 122, 269-278. 20. Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Thomason, L. C., Stahl, F. W. & Stahl, M. M. (1989) Genome 31, 53-67. 21. Kobayashi, I. (1992) Adv. Biophys. 28, in press. 22. Silverstein, Z. & Cohen, A. (1987) J. Bacteriol. 169, 3131-3137. 23. Birge, E. A. & Low, K. B. (1974) J. Mol. Biol. 83, 447-457. 24. Lloyd, R. G., Evans, N. P. & Buckman, C. (1987) Mol. Gen. Genet. 209,
135-141. 25. Lloyd, R. G. & Thomas, A. (1984) Mol. Gen. Genet. 197, 328-336. 26. Yamamoto, K., Kusano, K., Takahashi, N. K., Yoshikura, H. & Kobayashi, I. (1992) Mol. Gen. Genet., in press. 27. Lovett, S. T. & Kolodner, R. D. (1989) Proc. Natl. Acad. Sci. USA 86,
2627-2631. 28. Fox, M. S. (1966) J. Gen. Physiol. 49 (Suppl.), 183-196. 29. Mahan, M. J. & Roth, J. R. (1989) Genetics 121, 433-443. 30. Off-Weaver, T. L., Szostak, J. W. & Rothstein, R. J. (1981) Proc. Natl. Acad. Sci. USA 79, 6354-6358. 31. Orr-Weaver, T. L. & Szostak, J. W. (1983) Proc. Natl. Acad. Sci. USA 80, 4417-4421. 32. Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. & Stahl, F. W. (1983) Cell 33, 2S-33. 33. Resnick, M. A. (1976) J. Theor. Biol. 59, 97-106. 34. Lin, F., Sperle, K. & Sternberg, N. (1984) Mol. Cell. Biol. 4, 1020-1034. 35. Chakrabarti, S. & Seidman, M. M. (1986) Mol. Cell. Biol. 6, 2520-2526.
