bacteriophage A recombination - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 87, pp. 2790-2794, April 1990 Genetics

Evidence for the double-strand break repair model of bacteriophage A recombination (homologous recombination/gene conversion/DNA repair/crossing-over/red genes)

NORIKO TAKAHASHI*t AND ICHIZO KOBAYASHI*tt *Department of Infectious Diseases Research, National Children's Medical Research Center, Tokyo 154, Japan; tDepartment of Bacteriology, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan; and tDepartment of Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108, Japan Communicated by Franklin W. Stahl, December 26, 1989

ogy), is rec' (A ki1335 cIts857 P3). The kil mutation was isolated by H. Greer (10). BIK1199 is a A-resistant mutant from M5361. BIK1189 is M5361 cured of A. The recAl strains DH1 and DH5 (11) and plasmid pIK43 were described previously (12). The recombination rate of DH1(pIK43) to Neo' cells is as low as 4 x 1i-0 per cell generation (12). pIK60 was made by cutting pIK43 with Sal I, filling in the ends with the Klenow fragment of DNA polymerase I, and ligating. pKEN51, a gift from K. Yamamoto (University of Tokyo), was made by cutting pIK43 with EcoRV, filling in, and ligating with a Sac I linker sequence. The plasmid pTP232 has both reda and redfp genes repressed by the product of the lacI gene on the same plasmid (13). The plasmids pTP262 and pTP225 are similar to pTP232, but they carry reda alone and redfp alone, respectively (13, 14). AB1157 (= recd) strains carrying these plasmids were provided by A. Poteete (University of Massachusetts). Transformation by Rubidium. Competent cells were prepared by the rubidium method as described (11, 15), except that the cells were grown at 320C to a density of 108 per ml and then incubated at 440C for 15 min and at 370C for 1 hr. Aliquots (0.20 ml) ofthe concentrated competent cells (equivalent to 2.5 x 108 induced cells) in tubes were stored in liquid nitrogen. The cells were spread on L agar plates (with pH adjusted to 7.5 with Tris-HCI) containing kanamycin (50 ,ug/ml) or ampicillin (100 ,ug/ml) for overnight incubation at 320C. Analyses of the plasmids of the transformants were as described (12, 15, 16). Transformation by Electroporation. Cells containing the plasmids with red genes were grown in L broth with tetracycline (12.5 ,ug/ml) and isopropyl fB-D-thiogalactopyranoside (IPTG, 0.1 mM) for >2 hr and concentrated (17). Aliquots were stored frozen in liquid nitrogen. DNA was introduced into these cells (1-2 x 109) by a Gene Pulser (Bio-Rad) at 25 AF, 2.5 kV, and 200 £1 (17). The cells were grown in SOC medium (11) at 370C for 1 hr and then spread on the selective agar plates.

ABSTRACT We have obtained evidence for the repair of double-strand gaps promoted by the Red function of bacteriophage A. A double-strand gap was made in one of the two regions of homology in an inverted orientation on a plasmid DNA molecule. The gapped plasmid was introduced into Escherichwa coli cells expressing the reda (exo) and red(3 (bet) genes of A. The gap was repaired by DNA synthesis copying an intact duplex. This gap repair was sometimes accompanied by reciprocal recombination (crossing over). The gap stimulated recombination about 100-fold. Our results are compatible with previous proposals that A homologous recombination involves the following early steps: (i) generation of double-stranded ends by the packaging machinery or by the replication machinery; (ii) production of a single-stranded tail with a 3'-hydroxyl end by 5' -*3' degradation by A exonuclease (reda gene product); (iii) pairing of the single-stranded tail with a complementary strand from a homologous duplex with the help of i8 protein (redfi gene product); (iv) priming of DNA synthesis at this 3'-hydroxyl end to copy the second DNA molecule.

In the homologous recombination (Red) system of bacteriophage A, cos (cohesive end site), the site to be cleaved during packaging, turned out to be one of the "hot spots" of recombination (1). Recombination events are concentrated at cos among unreplicated A chromosomes, whereas they are distributed more or less uniformly along replicated A chromosomes (2-4). The "break-copy" model (5) once provided explanations for these features of A recombination (6). In contradiction to the prediction of this model, however, a cos site that could not be used to finish packaging a particular recombinant was shown to enhance formation of that recombinant (1, 7). An alternative to the break-copy model is that a double-strand break made at cos initiates, rather than terminates, recombination (1). When replication is allowed, the double-stranded ends associated with "rolling-circle" replication may initiate recombination in a similar fashion all over the chromosome. In the yeast Saccharomyces, recombination between a DNA segment with a double-strand gap and a homologous DNA segment was carried out in vegetatively growing cells (8, 9). The double-strand gap had been repaired by DNA whose sequence was derived from the intact homologue. We hypothesized that A's Red system may be able to effect a similar double-strand gap repair. Here we provide evidence supporting that possibility.

RESULTS Detection of Double-Strand Gap Repair. We examined plasmid recombination by A function. The design in Fig. 1 should enable us to recover both products of recombination, based on the assumption that gap repair will be intramolecular. Our substrate plasmid (pIK43) carries two deletion alleles of the neo gene in an inverted orientation. Because a linker sequence for Xho I is inserted at the upper deletion, cleavage with Xho I generates a double-strand gap corresponding to the deletion. The two products in Fig. 1B should be produced if intramolecular gene conversion by the lower, wild-type segment repairs this gap. These products carry a

MATERIALS AND METHODS Strains and Plasmids. M5361, an Escherichia coli strain provided by E. Signer (Massachusetts Institute of TechnolThe 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: Amp , ampicillin-rsitat Kn, kanamycinresistant; IPTG, isopropyl f3-D-thiogalactopyranoside. 2790

Genetics: Takahashi and

Kobayashi

A

Proc. NatL Acad. Sci. USA 87 (1990) B

A

E~~~ D

pIK43 X

h

o I~ho

0-

AP+

A

gene conversion without crossing-over (typeS)

gene conversion with crossing-over

(type6)

FIG. 1. (A) pIK43, the substrate plasmid. The neo gene is from TnS and makes the host cell resistant to kanamycin. The top segment has a deletion (a) between the two Nae I sites, which removed one end (the C end) of the neo gene. The Nae I site was inactivated by insertion of an Xho I linker sequence, 5'-CCTCGAGG-3'. The bottom sequence has a 248-base-pair deletion (b), which removed the other end of the neo gene. The left part (2321 base pairs), including the amp (ampicillin-resistance) gene and the replication origin is derived from pBR322. The entire length is 14,795 base pairs. The restriction enzymes (and their site coordinates) are as follows: X, Xho 1 (1082); N, Nae 1 (11109, 11392, 12590, 12750); RI, EcoRI (8024, 14793); RV, EcoRV (10367). (B) Double-strand gap repair by intramolecular gene conversion. Cutting pIK43 with Xho I makes a double-strand gap in the upper segment. The gap is repaired by copying the homologous sequence of the lower segment. This repair event restores a neo+ gene and can be selected for by kanamycin.

functional neo+ gene and therefore can be selected by kanamycin. We used an E. coli strain that carries bacteriophage A as prophage to provide recombination function. The prophage carries a gene encoding a thermolabile repressor (clts857), as well as mutations preventing cell killing (kl33S and P3). The A recombination function can thus be expressed by heating the cells without destroying their colony-forming ability. We introduced the gapped plasmid by the rubidium method into these cells expressing A function and then selected for cells carrying neo+ plasmids on kanamycin agar. Generation of the double-strand gap with Xho I increased kanamycin-resistant (KanR) transformants about 100-fold (Table 1). This increase

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FIG. 2. (A) Double-strand gap repair at various DNA inputs. A fixed amount of the competent cells (equivalent to 2.5 x 108 induced cells) of strain BIK1199 frec' (A)] was incubated with various amounts ofpIK43 (either intact or cut withXho I). The transformants (AmpR or KanR) were selected on agar. (B) Development of repair capacity in the cell after heat induction. The reck (A) cells (BIK1199) grown at 32TC were heated at 440C for 15 min. After various times of incubation at 3TC, the cells were harvested for preparation of the competent cells. pIK43, either intact or cut with Xho I, was introduced into these competent cells. The symbols in parentheses indicate finding of no colony at particular dilutions. For example, a symbol in parentheses at 10 means that no colony was detected when 1/10th of the sample was assayed. A, No cut, KanR; e, cut, KanR; A, no cut, AmpR; 0, cut, AmpR.

site on the boundary between the repeated sequence and the unique sequence (Fig. 1A). The increase in KanR transformants was observed over a wide range of DNA inputs (Fig. 2A). Restriction enzyme analysis showed that all 30 of the examined KanR transformants obtained by introduction of the gapped plasmid carried plasmids predicted by doublestrand gap repair effected by homologous recombination (Fig. 3; Table 2). The Xho I site that was present in the upper segment (Fig. lA) is no longer present (Fig. 3). Instead, the two Nae I sites, which flank the deleted region and were absent in the upper segment (Fig. lA), have been regenerated in the upper segment (Fig. 3). This demonstrates that repair of the double-strand gap has occurred by gene conversion. Analysis with EcoRI (Fig. 3) and Bgi II (data not shown) revealed that the plasmids in 15 of the 30 clones were indistinguishable from the starting plasmid, pIK43, except for the gene conversion at the gap (Table 2). This type of plasmid No cut Xhol EcoRV

Nae I

EcoR.I - U,

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= w A-

was not observed with a double-strand break by EcoRV at a Table 1. Plasmid double-strand gap repair by A function Condition Colony-forming units KanR AmpR Recipient Enzyme A+ None 780, 800 1, 0 A+ 80, 45 Xho I 81, 51 A+ EcoRV 0, 0