VIROLOGY

182, 316-323

(1991)

Bacteriophage ANTHONY Department

of Molecular

P22 Accessory

Recombination

Function

R. POTEETE,’ ANITA C. FENTON, AND ARLENE V. SEMERJIAN Genetics and Microbiology, Received December

University

of Massachusetts,

3 1, 1990; accepted

February

Worcester,

Massachusetts

0 1655

1, 199 1

The accessory recombination function (a@ gene of bacteriophage P22 is located immediately upstream of the essential recombination function (efl gene. Three mutant alleles of arfwere constructed and installed in P22 in place of the wild-type allele: an out-of-frame internal deletion, an in-frame internal deletion, and an amber mutation. The deletion mutant phages are partially defective in homologous recombination and plaque formation in wild-type and recA hosts; their defects are more severe in rec6 and recA rec6 hosts. The amber mutant phage exhibits the same growth phenotypes in nonsuppressing hosts, but not in an amber-suppressor host. Plasmids that express arfcomplement the growth defect of arf phages. These plasmids stimulate erf-mediated recombination; they were also found to cause a small stimulation of recA-recBCD-mediated homologous recombination of phage X. o 1991 Academic PRXS, IIIC.

INTRODUCTION

system and, to a lesser extent, recombination mediated by the host system. The results of these studies clarify relationships among the host and phage recombination-promoting proteins that are present in a P22infected cell.

Like other large, double-stranded DNA-containing bacteriophages, P22 encodes its own homologous recombination functions, and is thus only partially dependent on the host cell ret genes for this activity. Studies of the homologous recombination-related genes of P22 have so far led to identification and characterization of three: erf(essential recombination function, essential in a recA host for circularization of the linear DNA, a prerequisite for rolling circle replication; Botstein and Matz, 1970), abc1, and abc2 (antirecBCD; Fenton and Poteete, 1984; Murphy et al., 1987b). In addition, a fourth gene was identified and tentatively characterized as recombination-related by Semerjian et a/. (1989). That gene, arf (accessory recombination function) is the subject of this paper. Studies by Semerjian et a/. (1989) showed that a trans-acting function encoded by a small segment of DNA immediately upstream of erfin the P22 fL operon was required for growth of certain mutant strains of the phage. The requirement of the mutant phages for this function could be met by plasmids that express the red genes of phage X, suggesting that the missing function might have a role in homologous recombination. The DNA sequence upstream of erf revealed a candidate open reading frame, potentially capable of encoding an acidic polypeptide of 47 amino acid residues, with a calculated net charge of -1 1 at neutral pH. Studies described below show that At-f protein stimulates homologous recombination mediated by the P22

MATERIALS AND METHODS Bacteria Escherichia co/i. AB 1 157 (argE3 his-4 leuB6 proA thr- 1 ara- 14 galK2 lacy 1 mtl- 1 xyl-5 thi- 1 rpsL3 1 tsx-33 supE44) and the otherwise isogenic strain JC5547 (recA 13 recB2 1 recC22) were obtained from A. J. Clark. Strain W3110 /a@ L8 (Brent and Ptashne, 1981) was used for propagation of plasmids. Salmonella typhimurium LT2. Strains MS1 868 (leuAam4 14 r-m- sup”) and MS1 883 (leuAam4 14 r-msupE) were obtained from M. Susskind; the otherwise isogenic strains TP13 (sr/::Tn 10 recA), TP134 (recA), and TP198 (recB::TnlO) have been described (Poteete, 1982; Fenton and Poteete, 1984). A tetracycline-sensitive derivative of TPl98, designated TP333, was obtained by selection for growth in the presence of fusaric acid as described by Bochner et al. (1980). Strain TP336 (recA recB) was constructed by transduction of TP333 with P22 HTgrown on TP13, followed by selection of a sr/::Tn 10 recA transductant, and subsequent fusaric acid selection of a tetracycline-sensitive derivative.

Plasmids The construction of a series of plasmids in which various P22 and X genes are fused to P,a,UV5 and inserted into the lacl-expressing, tetracycline resistance-

’ To whom correspondence should be addressed at the Department of Molecular Genetics and Microbiology, University of Massachusetts Medical Center, 55 Lake Ave. N., Worcester, MA 01655. 0042.6822/91

$3.00

CopyrIght 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

316

P22 ACCESSORY

RECOMBINATION

conferring vector pMC7 has been described. These plasmids include pTP184, pTP224, pTP225, pTP232, and pTP262 (Fenton and Poteete, 1984; Poteete and Fenton, 1984; Murphy eta/., 1987b). An ad-expressing plasmid of this type, pAS470, was constructed by digesting pAS438 (Semerjian et al., 1989) with Rsal, ligating EcoRl linkers to the Rsal ends, digesting the resulting mixture with EcoRI, purifying the ca. 370-bp art-containing fragment thus produced, and ligating it into the fcoRl site of pMC7. A related series of plasmids, in which erfand arf are fused to the strong, lacl-regulated promoter PraC, and placed in pMC7, was constructed for these studies. pTP531 (erf only): The large, origin-containing fragment of pAS439 (Semerjian et al., 1989) generated by digestion with HindIll, filling in the HindIll ends with DNA polymerase I and dNTPs, and subsequent digestion with Pstl, was joined to the P,,-containing PstPvull fragment from ptacl2 (Amann et al., 1983) to form an intermediate, pTP503; this plasmid was digested with BarnHI, and the resulting ends were filled in and ligated with fcoRl linkers; digestion with fcoRl then released a ca. 960-bp P,,,- and erf-containing fragment that was purified and inserted into pMC7. pTP536 (arf + erf): approximately 35 bp of P22 DNA upstream of arf was removed by digesting pAS438 with HindIll and Ba131 exonuclease and then religating in the presence of Hindlll linkers to generate an intermediate, pAS499; the HindIll-BarnHI DNA segment containing arf and erf was fused to Pr,, and inserted into pMC7 as in the case of pTP53 1. pTP570 (arf only): this plasmid has the same structure as pTP536, except that the arf- and erf-containing segment is truncated at the Rsal site in erf, and thus contains only 67 bp of erf. In all of the pMC7-derived plasmids used in these studies, except for pTP224, pTP225, and pTP262, which were not tested, the orientations of the promoter-containing segments with respect to the rest of the plasmid are the same. Mutant alleles of arf were constructed in plasmids and then introduced into P22 by recombination. A plasmid bearing an internal deletion in arf, pTP563, was constructed from two fragments: a 316-bp fragment from pTP146 (Fenton and Poteete, 1984) generated by cutting with Ddel, filling in, and then cutting with EcoRI, and the large, origin-containing fragment of pAS439, generated by cutting with HindIll, filling in, and cutting with fcoRI. /-/indIll linkers (5’~CAAGCTTG3’) were added to the ligation, and two were incorporated between the two blunt ends in the resulting plasmid. A version of the deletion-bearing plasmid with an in-frame arf deletion, pTP568, was created by partial digestion of pTP563 with HindIll, filling in, and religating; in this case, only one of the two HindIll sites in

317

FUNCTION

pTP563 was cut (see the legend of Fig. 2). An amber allele of arf was generated by installing a 2-kbp arf-, erf-, and abc-containing fcoRl fragment from pTP147 (Fenton and Poteete, 1984) into the fcoRl site of Ml 3 mp18 and employing the oligonucleotide-directed mutagenesis procedure of Kunkel et al. (1987); the mutagenic oligonucleotide was 5’-AATCATGIAGCATGA-3’ (mismatch underlined). Following verification by DNA sequencing, the amber mutant fragment was recloned into pMC7 to generate plasmid pTP592. Phage Mutant alleles of arfwere installed in P22 by crossing the relevant plasmid with P22 erf-amH1046 cl-7 13-h21 (Murphy et a/., 1987a) and plating the progeny phage on a recA host to select for erf+. The presence of mutant arf alleles in the recombinants was confirmed by DNA sequencing. The out-of-frame arf deletion allele is designated arf-A563, the in-frame deletion arf-A568, and the amber allele, arf-am2 The deletion alleles were combined with 13-amHlO1 and 19amH447 (codons 20 and 113 of the respective genes -Rennell and Poteete, 1985 and unpublished observations) by standard phage crosses. XRFLP363 cl857ind S-am7 and X RFLP365 cl857ind R-am5 have been described (Poteete et a/., 1988). P22 and X crosses to measure recombination frequencies were carried out as described previously (Fenton and Poteete, 1984; Poteete et a/., 1988). In all of the experiments shown, crosses were done in triplicate; the recombination frequencies reported represent means, with standard errors indicated. RESULTS Constructions

involving

the atf gene

P22 mutants specifically deficient in arf function were constructed as described under Materials and Methods. Three alleles were constructed: an out-offrame internal deletion, arf-A563, an in-frame internal deletion, aff-A568, and an amber, a&am,?, in which the second codon of the putative arf coding region is replaced by TAG. A map of the arf region of the P22 chromosome, with the positions of the arf deletions indicated underneath, is shown in Fig. 1; the sequences of the arf gene and its mutant alleles are indicated in Fig. 2. The arf and erf genes, singly and in combination, were placed under control of lacl and Pr,, in plasmids constructed as described under Materials and Methods. The structures of these plasmids are indicated in Fig. 1.

POTEETE, FENTON,

318

AND SEMERJIAN

Sau96

TABLE 1 kil

0

t

PLAQUE FORMATION BY arf MUTANTS

Phageb

A563. AS68

c c

HostC wild-type

pTP536

FIG. 1. Map of the P22 chromosome in the vicinity of the erfgene. Sequences missing from the arf deletions are indicated by open boxes. Sequences present in the arf- and erf-expressing plasmids constructed for these studies are indicated by solid lines underneath: arrows represent Ptac. The Sau96 site in the phage has been converted to a BamHl site in the constructions described in the text by cutting, filling in, and ligating to a linker.

Growth and recombination phenotypes of P22 arfmutants P22 strains bearing mutant arfalleles were tested for their plaque-forming properties on a number of Salmonella hosts; wild-type and erf-am strains were included as controls. The results are shown in Table 1. In the wild-type host, a deficiency in &function reduces plaque size, indicating that arfis useful, but not essential, for growth; the same observation applies to the erf gene, although the growth defect of P22 erf-am is slightly greater than that of the P22 arf mutants. The growth defect of the arf mutants can be complemented by plasmids that express arf or the combination of X redo and reda, as observed previously (Semerjian et a/., 1989). Growth of the arf mutants is affected little, if at all, by expression of redor (A exonuclease), while it is inhibited by expression of either redp or X gam alone; in contrast, P22 erf-am is specifically complemented by

Met Gin His Glu Phe Ser Asp Glu Glu Phe Ile Ala ATG CAG CAT GAA TTT AGT GAC GAA GAA TTT ATC GCG T Leu Ile Ser Pro Glu Ile Glu Glu Glu Val Glu Gln CTT ATC TCT CCT GAA ATT GAG GAA GAG GTT GAG CAA Gln Ile Asn Leu Ala CAA ATC WC! TTA

Ala

Ile Glu Trp Asp AXI! GGT TGG

Phe Ala Gly Tyr Tyr Ser TTT GCG GGG TAT TAC TCA TGA

Glu

Glu

wt

A563

A568

am2

erf-am

++ ++

t t

t t

t t

flt/-

++

+t

t+

tt

t/-

++

tt

t

t

+t

++

++

+t

++

+t

++

-

-

-

tt

++

t/-

t

+

-

++

++

++

tt

++

++

t/-

i-l-

t/-

t/-

+t t+ tt tt

+ t/t/tt-

+ t/+ t

++ t/-

tt

t

-

tt

-

-

-

-

tt ++

-

t/-

-

-

+I-

+/-

++

t+

tt

+t

-

tt

t/-

t/-

+I-

tt

pTP570 pTP531

c

Plasmid (function)

Arg Gln Asn Pro Ile CGG 6BA AAT CCG ATT

FIG. 2. Sequence of the arfgene. The gene is shown reading left to right, reversing the orientation of Fig. 1. Sequences missing in the deletion alleles are underlined; these are replaced by C AAG CTT GCA AGC TTG AGC TTG G in the case of A563 (out-of-frame), and by C AAG CTA GCT TGC AAG Cl’T GAG CTT GG in the case of A568 (in-frame). The sequence of arf-am2-C to T in codon 2-is indicated as well.

supE recB recA

recA recB

none pMC7 (vector) pTP570 (arf) pTP53 1 (e4 pTP536 (arf + erf) pTP225 ( 1 redo) pTP262 (A reda) pTP232 (P + 4 pTP224 (A gam) none none none pMC7 (vector) pTP224 (1 mm) none pMC7 (vector) pPT570 brf) pPT531 (erf )

t

a Host cells were grown in LB (supplemented with tetracycline at 10 pglml in the case of plasmid-bearing strains) to a density of 2 x 10Blml. Portions of the cultures (0.2 ml) were mixed with 2.5 ml molten soft agar and spread on LB agar plates (supplemented with tetracycline at 10 pglml and 1 mM isopropyl fl-o-thiogalactopyranoside in the case of plasmid-bearing strains). Seven-microliter portions of the indicated phage suspensions, at 1O3 and 1 051ml, were spotted on the lawns; plaque formation was scored after overnight incubation at 30”. Key: ++, large plaques; +, small plaques; +I-, tiny plaques, reduced efficiency of plating; -, no plaques. b All phages carried, in addition to the indicated allele, cl-7 and 73-h2 1. The erf allele tested was erf-amH 1046. c Wild-type, MS1 868; supE, MS1 883; recA, TP134; recB, TP333; recA recB, TP336.

redp, as previously reported (Poteete and Fenton, 1984). In the wild-type (ret’) host, the three arf mutants differ little, if at all, in plaque-forming ability; however, the presence of a glutamine-inserting amber suppressor (supE; Winston et al., 1979) in the host strain suppresses the growth defects of phages bearing amber mutations-but not deletions-in arf. This lat-

P22 ACCESSORY

RECOMBINATION

ter observation suggests strongly that arf encodes a polypeptide. The plaque-forming abilities of arfmutants on a recf3 host are indicated in Table 1. Like erf mutants, they grow more poorly on the recB host than on wild-type; this property distinguishes them from abc mutants, which grow better on recB than on wild-type. In tests of plaque formation, as well as in other experiments described below, overexpression of erffrom a plasmid partially complements the growth and recombination defects of P22 arf mutants. This observation raises the question of whether arf has a distinct function in recombination or whether it might simply be required for efficient expression of erf. This latter hypothesis was tested by plating P22 arf mutants on a recA host. If the growth defect of arf mutants results entirely from a deficiency in erf expression, then it should become more pronounced in a recA host, in which P22 has a stringent requirement for erf. As shown in Table 1, this is not the case: P22 arf- grows about equally well in wild-type and recA hosts. Only the phage bearing an out-of-frame arf deletion exhibits a slightly enhanced recA dependence; this enhanced dependence might be attributable to a mild polarity effect resulting from frameshifting in translation. As in the wild-type host, X gam expression inhibits growth of the arf mutants in the recA host. The phenotypic distinctness of atfand erf mutants is further illustrated in their plating properties on a recA reck host. Neither type of mutant phage forms plaques on this host. The arf mutants are efficiently complemented by the arf-expressing plasmid, but not by the erf-expressing plasmid (except for a small stimulation of the out-of-frame deletion mutant, again possibly due to relief of polarity on the phage-borne erf gene). The erf mutant, as expected, displays the opposite pattern of complementation. The data in Table 1 indicate that the presence of the vector plasmid in the recA recB host exerts a small stimulatory effect on plaque formation by arf mutants. This effect is also observed in the recB host (not shown), but not in the wild-type host. Results presented above, as well as earlier observations (Semerjian et al., 1989) suggest that arf is a component of the P22 homologous recombination system. To explore this possibility, we constructed P22 strains bearing arf deletion alleles in combination with amber alleles in genes 13 and 19, and measured production of am+ progeny in crosses; arf+ phages were crossed at the same time for comparison. The interval in which recombination can produce am+ progeny in these crosses is approximately 590 bp in length, and located approximately 10,300 bp away from the arf gene (in

319

FUNCTION

Wild

Type

recA

r&Z8

FIG. 3. Recombination defects of P22 arf mutants. Crosses between P22 13-amHlO1 cl-7 and P22 14amH447 ~1-7, in which each phage bore wild-type (diagonally striped bars), A563 (open bars), or A568 (checked bars) alleles of arf, were carried out as described previously (Fenton and Poteete, 1983). Values indicated are two times the percentage of am+ phage among the progeny of the crosses, wrth positive standard errors shown above the bars.

one direction around the circular map-farther in the other direction). Results shown in Fig. 3 indicate that, in a wild-type host, arf mutants recombine less than wild-type P22. In a recA host, wild-type P22 recombines less than in a wild-type host, as shown previously (Botstein and Matz, 1970; Fenton and Poteete, 1984); in the recA host, a lack of arf function decreases the recombination frequencies only slightly. In a recB host, recombination by wild-type P22 is similarly decreased; in this case, however, the recombination deficiency of arf mutants relative to wild-type appears to be even greater than in the wild-type host. Crosses between arf mutant phages in the recB host result in the lowest recombination frequencies seen in these experiments. The experiment summarized in Fig. 4 indicates that the effects of plasmid-expressed phage recombination functions on recombination by arf mutant phages largely parallel the effects of these functions on plaque formation by the arf mutants. Thus, both arf and erf complement the arf mutant phage; the phenomenon whereby overexpression of erfcompensates for lack of arf in the wild-type host is particularly apparent in this experiment. The combination of X red6 and redor stimulates recombination by the arf mutant, while redcv by itself has no effect, and red/3 and gam inhibit it. Recombination by an at-f mutant in a recA recB host was examined in the experiment shown in Fig. 5. As in the wild-type host, the arfmutant has a recombination deficiency, which is remedied by expressing arffrom a plasmid. In the recA recB host, though, overexpression of erfdoes not compensate for a lack of arf. The degree of recombination proficiency exhibited by the arf mutants in various hosts, and in the presence of various

320

POTEETE, FENTON,

AND SEMERJIAN

5

0.15

E .d i

0.10

kz *

0.00

v NO plasm10

pTP224 lgaml

pTP225 (red II

pTP232 II t aI

pTP262 pTP531 Ire0 11 IWfl

pAS470 bfl

FIG. 4. Effects of plasmid-encoded phage recombination functions on recombination by a P22 arf mutant. Crosses and data presentation were as described in the legend to Fig. 3. Expression of plasmidborne recombination genes was induced by addition of IPTG to 1 mM, 30 min before infection. Diagonally striped bars, wild-type; open bars, = arfiA563.

phage recombination functions, largely parallels the plaque-forming capability of the mutants in the same situations. We conclude that arf function is essential for full activity of the P22 homologous recombination system; it is likely that the contribution that arf makes to growth of the phage resides in this role. Stimulation

0.05

of recombination

by arf

To explore further the role of arf in homologous recombination, we conducted crosses with derivatives of phage X, called X RFLP (Poteete et al., 1988) that lack their own recombination systems. X RFLP, unlike comparable derivatives of phage P22, grows well in wild-type and recA recBC hosts. In the recA recBC host, X RFLP experiences very little recombination, which can be greatly stimulated by phage recombina-

pMC7

IControl)

pTP531 (et-f)

pTP570 (art)

FIG. 5. Recombination defect of a P22 arf mutant in a recA recB host. Crosses and data presentation were as described in the legend to Fig. 3. Diagonally striped bars, wild-type; open bars, arf-A568.

pHC7 lcontroll

pAS470 larf)

pTP570 brf)

pTP531 lerfl

pTP536 lerf

+ arf)

FIG. 6. Stimulation of erf-mediated recombination by arfin a recA recB recC host. Crosses between X RFLP363 c1857ind S-am7 and XRFLP365 cl857ind R-am5 were carried out as described previously (Poteete et a/., 1988) with IPTG added to 1 mAY, 30 min before infection. Samples of the cultures were taken immediately before infection, lysed by heating in SDS-containing buffer, and subjected to SDS-polyacr-ylamide gel electrophoresis. The gel was stained with Coomassie blue, and the visible Erf protein bands in samples of pTP531- and pTP536-bearing plasmids were quantitated by scanning densitometry and normalized to a solitary host band of compable intensity. Relative amounts of Erf present in the cells determined in this way were 1 .O and 0.99 for the two plasmids, respectively.

tion genes expressed from plasmids (unpublished observations). In these crosses, recombination is measured in the interval between S-am7 and R-am5 (analogous to the 13-19 interval measured in the P22 crosses), by the production of am+ progeny. As shown in Fig. 6, arfdoes not stimulate recombination by itself, while erf does. The combination of arf and erf is far more effective than erf alone. In these experiments, it was possible to measure erf expression by scanning densitometry of Coomassie blue-stained SDS-polyacrylamide gels run with samples made from the infected cells. The cells bearing the arf + erf-producing plasmid did not contain more Et-f protein than the erfonly plasmid-bearing cells (see the legend to Fig. 6). Stimulation of erf-mediated recombination by arfin this case thus cannot be simply a consequence of an effect of arf on erf expression. The results of experiments described above raise the interesting possibility that arfis a general recombination-enhancing function, able to stimulate recA-mediated recombination as well as that mediated by erf. The argument for this interpretation is as follows: recA function contributes substantially to P22 recombination in a wild-type host (Fig. 3). If arf stimulated only erf-mediated recombination, then the recombination deficiency of an arf mutant should be worse in a recA host than in wild-type, because, in the recA host, P22 would be completely dependent on erffor recombina-

P22 ACCESSORY

RECOMBINATION

FUNCTION

321

arf has been observed: a 1O-fold inhibition of recombinant formation in a reck recipient (not shown).

DISCUSSION

pMC7 IControll

pAS470 bfl

pTP570

pTP531

IWfl

(Wfl

pTP536 lerf

+ arf)

FIG. 7. Stimulation of wild-type host system-mediated recombination by &and eff. Crosses and data presentation were as described in the legend to Fig. 6. Plasmids pAS470 and pTP570 bear f,,, and P,, respectively, fused to the arf gene. Data presentation was as described in the legend to Fig. 3.

tion. This appears not to be the case, however: as shown in Fig. 3, the recombination deficiency of arf mutants relative to wild-type phage is no worse in the recA host than in the wild-type host. The simplest interpretation of this observation is that &can stimulate recA-mediated recombination. Stimulation of recA-mediated recombination by arf was assayed directly in the experiment shown in Fig. 7. In this experiment, X RFLP phages were crossed in a wild-type host, and the effects of arfand erf(expressed from plasmids) on the frequency of recombination in the S-R interval were measured. In this system, in the absence of erf, arf causes a small (2-fold in this representative case; up to 2.5-fold in other experimentsnot shown) increase in the frequency of recombination mediated by the wild-type host system. The degree of stimulation depends on the level of expression of arf: expression of arf under control of P,,, instead of the stronger P,, gives a smaller stimulation (generally not statistically significant). As shown in the figure, expression of erf in the wild-type host also results in a higher level of recombination-as might be expected, since erf promotes recombination even in the absence of host ret functions. The combination of arf and erf is still more effective. The finding that arfcan stimulate X recombination by the E. co/i system led us to test whether it could also stimulate formation of recombinants following Hfr crosses. Wild-type cells bearing arf-expressing and control plasmids were used as recipients in mating experiments carried out as described previously (Poteete and Volkert, 1988). No significant stimulation of recombinant formation by arf was detected (not shown). In such mating experiments, only one significant effect of

These studies lend support to the notion that the arf gene of P22 encodes a small polypeptide that stimulates phage recombination, as hypothesized previously (Semerjian et a/., 1989). Evidently, the Arf protein is a general recombination enhancer, capable of stimulating not only E&mediated, but host system-mediated recombination as well. In this latter property, Arf is reminiscent of the Ref function of phage Pl (Windle and Hays, 1986; Laufer et al., 1989) although its observed stimulatory effects have been limited to recombination between phage chromosomes. The small size and acidic amino acid composition of Arf led to the hypothesis that it might function as a DNA analog in strand exchange reactions, perhaps accelerating unloading of ssDNA from binding sites in the Erf protein into dsDNA (Semerjian et al., 1989). The present studies suggest that Arf might perform the same function in RecA-DNA or Red@DNA interactions. These hypotheses remain to be tested by biochemical experiments.

Functional roles of the P22 recombination proteins Genetic and biochemical studies of homologous recombination in phage P22 have so far uncovered four protein species that appear to contribute to the process: Et-f, RecA, Abe-RecBCD, and Arf (Botstein and Matz, 1970; Fenton and Poteete, 1984; Poteete et al., 1988; this paper). While the contributions of RecA, Et-f, and Arf are all readily apparent from the phenotypes of null mutations in the respective genes, the contribution of Abe-RecBCD complex to recombination is less so. The P22 Abel and Abc2 proteins both modulate RecBCD activities (Murphy eta/., 1987b; Poteete eta/., 1988); Abc2 has been shown to form a complex with RecBCD (K. C. Murphy, personal communication). However, relative to wild-type, P22 mutants lacking Abe function demonstrate little or no deficiency in homologous recombination in the wild-type host. Two new observations concerning Arf function, though, highlight the role of Abe-modulated RecBCD in P22 recombination: (1) Arf is capable of stimulating the host cell recombination system, even in the absence of Erf, Abe, or Red proteins. (2) The growth and recombination deficiencies of P22 arf mutants are exacerbated in a recB mutant host (as well as in a wild-type host in which RecBCD has been inactivated by X Gam protein). Thus, A&enhanced recombination depends on RecBCD, which, in a P22-infected cell, exists as a complex with Abe. The Abe proteins’ function can be

322

POTEETE, FENTON, AND SEMERJIAN A •-I

exo

,

bet

H gam 5

&

sieB4

kil pL

P22

FIG. 8. PL operons of P22 and X. A break has been introduced in the X map so that regions of high homology, indicated by bold lines, are aligned. Gene boundaries and identifications are taken from lneichen et al. (1981), Sanger et a/. (1982), Semerjian et a/. (1989), and K. Ranade and Poteete (unpublished data).

understood as one of modulating RecBCD to make it less prone to interfering in phage replication, but still available for phage recombination. Relationships of P22 and X recombination proteins P22 and X exhibit a conservation of genome structure far greater than the limited sequence homology of the two phages would suggest. Campbell and Botstein (1983) have proposed that the lambdoid phage chromosomes have evolved as groupings of interchangeable modules, each module consisting of a group of genes that carry out some particular function, such as lysis or recombination. Modules are linked by segments of high sequence homology, in which homologous recombination can exchange them between phages. This pattern of conservation can be seen in the partial maps of the two phages’ PL operons shown in Fig. 8. The homologous recombination systems of P22 and X constitute modules of the type envisioned by Campbell and Botstein; the two phages have been shown to recombine in vivo to produce hybrid X phages bearing the P22 recombination genes (Hilliker and Botstein, 1976). P22 lacking its own homologous recombination system can grow and recombine if supplied with the X system; similarly, X can make use of the P22 system (Poteete and Fenton, 1984). This functional interchangeability is not reflected in any apparent structural homology. Moreover, analogous individual parts of the two systems exhibit variable interchangeability: some combinations of hand P22 homologous recombination genes are functionally incompatible, for example Red@ + Erf + Abel + Abc2 (Table 1, Fig. 4). Et-fand Red@proteins are clearly analogous in function. Red/3 complements the growth and recombination defects of P22 erf mutants (Poteete and Fenton, 1984). Both proteins bind single-stranded DNA, and, under certain conditions, promote renaturation of ho-

mologous single strands in vitro (Kmiec and Holloman, 1981; Poteete and Fenton, 1983). It is reasonable to speculate that these proteins, like RecA, participate in homologous recombination at the level of strand exchange. The observation that Red@inhibits the growth and recombination of a P22 atfmutant, though, indicates that it is not completely interchangeable with Et-f. The simplest interpretation of the inhibition is that, unlike Erf, Red/I either cannot work in concert with AbcRecBCD, or can do so only in the presence of Ar-fprotein. [Normally, Reda works with Reda, the X exonuclease (Radding, 1970)]. The reasoning outlined above suggests that the P22 analog of the X exonuclease is Abe-RecBCD. This analogy is supported by the observation that the Abcmodulated RecBCD protein found in P22-infected cells, unlike the Gam-modulated RecBCD of X-infected cells, retains significant nuclease activity; this residual activity differs qualitatively from that of unmodified RecBCD (Poteete et al., 1988; K. C. Murphy, personal communication). Thus, P22 “hijacks” the host’s RecBCD nuclease for its recombination system, while X completely inactivates RecBCD, and substitutes its own nuclease. The foregoing discussion would appear to leave X Gam and P22 Arf proteins as analogs, in harmony with their map locations. This analogy is not compelled by experimental observations, which suggest instead that Gam and Abe are analogs. Gam protein complements a P22 abc mutant, while it actually interferes with growth and recombination of an arfmutant. Moreover, Gam and Abe proteins both act directly on RecBCD (Karu et a/., 1975; Poteete et a/., 1988; K. C. Murphy, personal communication). On the other hand, the Abe and Gam proteins modulate RecBCD in qualitatively different ways, and Gam protein can substitute successfully for Abe only if Ar-fis present. The relationship between the recombination-stimulating Arf protein and Gam, which, if anything, inhibits the host cell recombi-

P22 ACCESSORY

RECOMBINATION

nation system (Poteete et al., 1988), is clearly not simple. The Gam protein acts on the product of the sbcC gene as well as on RecBCD (Kulkarni and Stahl, 1989). In addition, we have observed that Gam protein stimulates recombination between X RFLP (red- gam-) phages in a recA recB recC host (unpublished observations). ACKNOWLEDGMENTS This research was supported by NIH Grant A118234. A.R.P. was supported by a research career development award from NIH.

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