Annealing Invasion in Phage A Recombination · ful repair of a double-strand end involves interac- tion with homologous DNA (RESNICK 1976, 1978; KRASIN and HUTCHINSON 1977). Such

Copright 8 1997 by the Genetics Society of America

Annealing us. Invasion in Phage A Recombination

Mary M. Lynn Thomason,* Anthony R. Poteete? Trudee Tarkowski,* Andrei Kuzminov* and Franklin W. Stahl*

*Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 and tUniversity of Massachusetts Medical School, Department of Molecular Genetics and Microbiology, Worcester, Massachusetts 01 605

Manuscript received April 28, 1997 Accepted for publication July 18, 1997

ABSTRACT Genetic recombination catalyzed by A’s Red pathway was studied in rec” and r e d mutant bacteria by

examining both intracellular A DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec” cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of R e d and the presence of non-allelic DNA ends, the Red pathway of A catalyzes recombination primarily by annealing.

D NA breakage generates double-strand ends. Faith- ful repair of a double-strand end involves interac-

tion with homologous DNA (RESNICK 1976, 1978; KRASIN and HUTCHINSON 1977). Such repair i n vivo is thought to proceed by two pathways. Both proposed pathways start with exonuclease-mediated processing of the double-strand end (Figure lB), which removes the 5‘ending strand creating a S‘ending single-strand overhang (Figure 1C). This single-strand overhang, with the help of synaptase, homologously pairs with and invades an intact duplex (Figure 1D). We will refer to this mechanism as “invasion” (Figure 1E). Synaptases catalyze homologous pairing of single-strand DNA with duplex DNA followed by strand exchange, generating a new duplex by displacing the homologous strand of the old duplex. The best studied synaptase is RecA protein of Escherichia coli and other bacteria (reviewed in KOWALG ZYKOWSKI and EGGLESTON 1994; ROCA and COX 1997). RecA homologues are found also in eukaryotes (reviewed in HEYER 1994). The invasion scheme predicts regions of hybrid DNA (hDNA) in which two strands are from different parents; the length of this hDNA may be variable with a mean length estimated at 1 kb (e.g., STAHL 1979).

Although RecA-catalyzed repair of double-strand ends via invasion is wide-spread, an alternative mechanism is available when each of two homologous duplexes has an end to be repaired (SZYBALSKI 1964; THOMAS 1966; THOMAS 1967). In this scheme, both

Corresponding authoc Franklin W. Stahl, Institute of Molecular Biol- ogy, University of Oregon, Eugene, OR 97403-1229. E-mail: [email protected]

‘Deceased January 22, 1996.

Genetics 147: 961-977 (November, 1997)

ends are processed by exonucleases, creating exposed complementary strands that can anneal (Figure 1, F and G) . Since annealing of complementary single strands is catalyzed by a variety of proteins that bind single- stranded DNA (reviewed in K ~ w ~ ~ c m o w s ~ ~ and EG GLEsTON 1994), the hallmark of this “annealing” mechanism is its independence of synaptase activities. Ac- cording to the model, the distance between the two interacting ends determines the extent of the hDNA (Figure 1G). Thus, if the distance is 10 or 20 kb, annealing may generate segments of hDNA longer than those arising by invasion.

Repair of a double-strand break by invasion or by annealing is recombinational. Indeed, much of our understanding of recombinational repair was gained through studies of genetic recombination per se. Phage A, a temperate bacteriophage of E. coli, has been useful in these studies for several reasons. (1) Both the phage and its host are exceptionally well studied. (2) Muta- tions have defined the principal recombination functions in both. (3) Recombination occurs at a rate high enough to study but low enough to correlate informa- tional and physical transfer between chromosomes. (4) Even though recombination is stimulated by DNA replication, crosses that are blocked for replication give a high frequency of recombinant offspring because dimerization, by recombination involving at least one circular chromosome, stimulates DNA packaging (reviewed in FEISS and BECKER 1983; THOMASON et al. 1997),

Soon after the discovery of the recA gene of E. coli (CLARK and MARGULIES 1965) it was found that A recombines with almost undiminished frequency in recA mutants (TAKANO 1966; VAN DE PUlTE et al. 1966;

962 M. M. Stahl et al.

BROOKS and CLARK 1967). The X genes reah and re@ are required for this RecA-independent X recombination (FRANKLIN 1967; ECHOLS and GINGERY 1968; SIGNER and WEIL 1968; SHULMAN et al. 1970); the corresponding proteins are an exonuclease (LITTLE 1967; SHULMAN et al. 1970; CARTER and RADDING 1971) and an annealing protein (KMIEC and HOLLOMAN 1981; MUNIYAPPA and RADDING 1986). Later, an adjacent gene, gam, was found to be required for maximal levels of Red-promoted recombination (ZISSLER et al. 1971; ENQUIST and SKALKA 19’73). Gam protein binds to the host RecBCD nuclease and inhibits all its known activities (UNGER and CLARK 1972; KARU et al. 1975; MURPHY 1991; but see SALAJ-SMIC et al. 1997), which would otherwise degrade the linear concatemeric products of X rolling-circle replication (ENQUIST and SKALKA 1973; GREENSTEIN and SKALKA 1975).

A proposed recombination route for X’s Red system, in keeping with the demonstrated in vitro properties of the Reda exonuclease, elaborates on the scheme of SZYBALSKI and THOMAS (CASSUTO and RADDING 1971) (Figure 1, C-G). The Reda protein is a 5’-3’ exonuclease acting on duplex DNA to expose 3‘ single- strand overhangs. If two homologous DNA molecules are degraded from overlapping double-strand ends, they may expose homologous segments of complementary single strands (Figure 1C). Complementary single strands can anneal with the help of RedP protein (Fig- ure 1F). “Strand assimilation,” followed by ligation, could complete the annealing reaction (CASSUTO and RADDING 1971; CASSUTO et al. 1971) (Figure 1G).

Later, the reintroduction (SZOSTAK et al. 1983) of double-strand break repair models for recombination (RESNICK 1976) and the subsequent finding that double-strand breaks stimulate X recombination in recA mutant cells prompted a proposal that X’s Red system, without assistance from RecA, catalyzes recombination by invasion (STAHL et al. 1985; STAHL and STAHL 1985; THALER et al. 1987). Experiments with plasmids were also interpreted to signify Red-promoted invasion, with Red@ protein acting as a synaptase (TAKAHASHI and KOBAYASHI 1990). However, no synaptase activity was found for RedP protein in vitro (MUNIYAPPA and RAD- DING 1986), questioning the validity of the later models. In fact, these later models slighted earlier in vivo studies that revealed qualitative differences in recombination of reo? X in the presence or the absence of RecA. Whereas X X X recombination is largely RecA-independent in standard crosses (BROOKS and CLARK 1967; FRANKLIN 1967), it is largely RecA-dependent when phage DNA replication is blocked (STAHL et al. 1972a,b, 19’74) This RecA-dependence is not due to the hypo- thetical destruction of Red recombination intermedi- ates in recA mutant cells by RecBCD nuclease (STAHL et al. 1978), contrary to the suggestion based on studies of the intracellular pool of X DNA (WILKINS and MISTRY

1974). Furthermore, other in vivo experiments indicate that when the Red system of h is asked to recombine linear with circular DNA, R e d is required (CLYMAN and BELFORT 1992; POTEETE and FENTON 1993). The mechanisms of X recombination catalyzed by the Red pathway in the presence or absence of RecA protein needed reevaluation.

The two models for X recombination (Figure 1) generate testable predictions. For example, the invasion scheme makes several predictions. (1) In the absence of DNA replication, X recombination depends on RecA (the only synaptase activity known to be available to X). (2) A solitary double-strand break is sufficient to stimulate recombination. (3) The track of hDNA formed at the site of invasion is relatively short. The last prediction appears to be in disagreement with the findings that some of the infrequent recombinants between close markers, arising in the presence of RecA, are hybrid along at least half the length of the X chromosome (RUSSO 19’73; WHITE and FOX 1975). However, such long segments of hDNA are rare among the abun- dant recombinants between distant markers, implying that recombinants between close markers are formed preferentially in long segments of hDNA (STAHL and STAHL 1976; HUISMAN and FOX 1986). By contrast, the annealing scheme predicts that (1) X recombination is independent of RecA, (2) recombination is possible only when non-allelic double-strand breaks are delivered to parental chromosomes, and (3) hDNA may span the entire length between the two double-strand ends. We tested these predictions using both physical and genetic techniques. Our results reveal the following. (1) In the absence of RecA protein, recombination of X promoted by the Red pathway is mostly (but not entirely) of the annealing type. (2) DNA replication provides favorable substrates for annealing. (3) In the presence of RecA, recombination is preferentially by invasion.

MATERIALS AND METHODS

General: Bacterial strains, phage strains and plasmids are in Table 1. Methods not detailed were as in ARBER et al. (1983). Cells harboring plasmids were grown with appropriate antibiotics (tetracycline at 10 ,ug/ml, ampicillin at 100 pg/ml).

Phage crosses and density gradient centrifugation: Except where indicated, both parental phages were density-labeled to verify the block to DNA replication and to eliminate unadsorbed phage from the analysis. Crosses were in light medium, and progeny phage particles were fractionated in cesium for- mate equilibrium density gradients. Density fractions were collected through a needle hole in the bottom of the tube and were assayed for plaque-forming units as described (STAHL et al. 1990).

Phage construction: Insertion of a XhoI linker into A’s SstII site to create SstII ::XhoI, right of CI at 40,389, destroyed the SstII site (allele X ~ O I ~ . ~ of HAGEMANN and ROSENBERG 1991). Insertion of an EcoRI linker into A’s unique, native XhoI site (33,498 bp from A’s left end) (X7zd::EcoRI) destroyed the

Genetic Recombination in Phage A

TABLE 1

E. coli strains, phages and plasmids

963

Designation Genotype/description Source/reference

E, coli strains 594 C600 K12SH-28 FA77 FZ14 FS1585 FS3809

MMS354 MMS429 MMS1833 MMS2228 MMS2480 MMS2488 MMS2554 MMS2622 MMS2539 MMS2597 MMS2660 MMS2661 MMS2662 MMS2663 MMS2666 MMS2735 MMS2737 MMS2739 MMS2740

pPAORM3.8 pTP645 pTP648 pTP666 pTP699

Phage X strains

Plasmids

Su- rec" SuII rec' SuII rec" Su- dnaBts22 Su- dnaBts22 recA56 SuII SuIII recD1009 594 A(~rlR-recA)306~

Jtsl5 int4 e1857 Psw80 int4 Psw80 Rts129 Btsl int4 cI26 Psw80 Btsl int4 cI26 Psus80 Ssw7 Jtsl5 SR1::XhoI int4 Xho1::EcoRI 13857 Psw80 SR1::XhoI int4 Xho1::EcoRI Psw80 Rts129 int4 Xho1::EcoRI Psw80 SstI1::XhoI Rh129

Jtsl5 int4 Xho1::EcoRI a 8 5 7 P s w 8 0 SstI1::XhoI ( int4 ?) Aspi6 Psw80 SstI1::XhoI gam210 cI857 Psw80 Anin5 Ssus7 SR3" cI857 Psw80 Anin5 SR3" Xho1::EcoRI cI857 Psw80 Anin5 int4 Aspi6 cI857 Psus80 int4 Aspi6 ~1857 Psw80 SstI1::XhoI (int4.P) Aspi6 cI857 Psw80 Anin5 Asus32 (int4B) Aspi6 cI857 SstI1::XhoI Asus32 Aspi6 cI857 Asus32 cI857 Anin5 Asus32 (int4B) Xho1::EcoRI cI857 Anin5

pBR322-based, Amp' Pa.& restriction system pMB9-based, Tc' cI w h re@ pMBSbased, Tc' cI pMB9-based, Tc' cI r e h re@ gam pMB9-based, Tc' GI, gam

WEIGLE (1966) APPLNARD (1954) FANGMAN and NOVICK (1966) MCMILIN and RUSSO (1972) STAHL et al. (1972b) STAHL et al. (1986) This study

Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection

GINGERAS and BROOKS (1983) POTEETE and FENTON (1993) POTEETE and FENTON (1993) POTEETE and FENTON (1993) POTEETE and FENTON (1993)

a FS1313 (lab collection) plated selecltively for Tc" (BOCHNER et al. 1980; WOY and NUNN 1981).

XhoI site. Insertion of a XhoI linker into X's leftmost EcoRI site (21,226 bp from X's left end) (SR1::XhoI) duplicated the EcoRI site. The alterations of the three restriction sites were conducted in vitro on small plasmids canying the relevant segment of A DNA. The plasmids were then picked up into X by homologous recombination. Cointegrate phage particles were selected by density (heavy) resulting from their in- creased DNA content. Phage particle segregants from the cointegrates were then isolated by density (light) and screened for the desired change at the restriction site.

Other genotypes were made by lytic cycle crosses between UV'd phage stocks.

Purification of phage in CsCl gradient: Phages used in physical assays were purified by banding in CsCl equilibrium density gradients with subsequent dialysis against TM buffer. A phage to be purified w a s plated in the evening and incubated at 32". The next morning, a single plaque was picked, placed in 1 ml of TM and kept at room temperature for at least 2 hr. Meanwhile, a fresh plating culture was prepared: a fresh overnight culture in TB was diluted 10-fold into TB [supplemented with the following per 100 ml: 200 p1 of 5 mg/ml B1, 500 pl of thymine (2 mg/ml), 500 p1 of 1 M

MgS04, 2 ml of 10% maltose], shaken for 2 hr at 37" and diluted twofold with TM. Twenty plates were poured (25 ml per plate) with the following additions per 400 ml of bottom Tryptone agar: 1.2 ml of 50% glucose, 160 pl of 0.01 M FeCls, 120 p1 of 0.25 M caCIp, 800 p1 of 5 mg/ml B1, 800 pl of 1 M MgS04. Plates were dried open for 30-40 min at room temperature. When the plates were ready, 6 ml of the fresh plating culture were mixed with 1 ml of TM with phage and incubated for 10 min at 37". Fifty-five milliliters of top Tryp tone agar was added, and the whole mixture was plated, 3 ml per plate, on the 20 plates. The plates were incubated at 37" until the plaques were touching each other (usually, 4-5 hr). The top agar was scraped from the plates and collected into a 250-ml centrifuge bottle. TM (100 ml) was added to the agar, and the suspension was left on ice for 2 hr with occa- sional mixing. The suspension was spun at 15,000 X gfor 20 min to sediment agar and unlysed cells. The supernatant was transferred to a fresh tube and left at 4" overnight. The next morning, the supernatant was spun at 15,000 X gfor 30 min to remove remaining traces of agar. The phage was then pel- leted at 28,000 rpm (100,000 X g) in an SW28 rotor for 90 min in a Beckman ultracentrifuge. After addition of 2 ml of

964 M. M. Stahl et ul.

TM, the pellet was left overnight at 4". Next morning, the pellet was resuspended gently with a pipette; 3.23 g of CsCl and TM to a total weight of 7.4 g were added, and the suspension was centrifuged in an SW50.1 rotor at 40,000 rpm (150,000 X g) for 20 hr in a Beckman ultracentrifuge. A blueish band of phage in the middle of the tube was removed with a pipette ("800 p1) and dialyzed against three changes of TM buffer in 250-fold excess. The titer of the phage thus purified is 2-8 X 10'2/ml.

Phage DNA isolation and anatysis: Overnight LB cultures of plasmidcontaining cells were diluted 100-fold into 20 ml of TB supplemented with antibiotics (see MATERIALS AND METHODS) and with 0.1% maltose and grown with shaking at 28" to 1-2 X 1Oscells/ml. For the last 30 min of incubation, IPTG was added to a concentration of 1 mM. Cells were sedimented by centrifugation, resuspended in 20 ml of TM sup plemented with 1 mM IPTG and incubated with shaking at 28" for 1 hr. After starvation, cells were collected by centrifugation and resuspended in l ml of TM. Cell density was determined by direct counting in a Petroff-Hausser chamber, and 5 X 10' to 2 X lo9 cells were mixed on ice in 200 p1 of TM with phages at an indicated multiplicity of infection. Phages were allowed to adsorb for 10-15 min, then the adsorption mixture was transferred to 37" for 1 min, after which the entire mixture was diluted into several ml LB broth pre- warmed at 37" and supplemented with 1 mM IPTG. The infected culture was then incubated with shaking at 37". One milliliter aliquots of the cultures were taken at intervals; the cells were sedimented in an Eppendorf centrifuge, resuspended in 100 pl of 1% SDS, 10 mM Tris HCl pH 8.0, 1 mM EDTA, and lysed by addition of 120 pl of phenol and vigorous vortexing. Phenol was removed after centrifugation; the aque- ous phase was extracted once with 120 pl of phenol/chloroform (1:l) and once with 120 pl of chloroform. Nucleic acids were precipitated twice with ethanol and resuspended in 100 p1 TE. One-third to one-half of each DNA preparation was used for restriction digestion and for a single electrophoretic separation. Each restriction reaction was in a total volume of 100 p1; NcoI was used to define the region of interest in phage DNA, while Nud and POuII, neither of which cuts in the region, were added to relocate signals originating from plasmids.

Electrophoresis and blot-hybridization: Electrophoresis was in 0.7% agarose in Tris-acetate buffer. Gels were soaked for 40 min in 0.2 M HCl, then for 45 min in 0.5 M KOH and finally for 15 min in 1 M Tris HC1 pH 8.0,1.5 M NaCl. DNA was then vacuum-transferred to Zeta-Bind membrane (CUNO), cross-linked to the membrane with UV, incubated in 5% SDS, 0.5 M sodium phosphate pH 7.4 for 30 min at 60" and hybrid- ized with a probe in the solution of the same composition for 10 hr at 60". Radioactive probes were prepared by random primer labeling. After hybridization, membranes were washed three times for 10 min in water, dried and either exposed to autoradiography film or directly scanned by AMBIS.

RESULTS

Red-promoted recombination is less RecAdepen- dent when non-allelic double-strand breaks are provided When DNA replication is blocked, A chromosome packaging, which is recombination-dependent, is dependent on the host RecA protein (STAHL et al. 1974, 1990). In crosses between unreplicating density-labeled phages, the recombinant and total phage yields of the

recA+ cross are several hundred times higher than the corresponding yields of the recA mutant cross (illustrated in Figure 2, A1 and Bl). Recombination is concentrated near the right end of the A chromosome, as revealed by the inheritance of the unselected cI+ marker, which lies between one selected marker to its left and another near A's right end. This nonrandom placement of exchanges (recombinational hotspot) reflects proximity to the cos site (STAHL et al. 1985). A phage-encoded enzyme, terminase, cleaves cos late in the infection. In crosses that are mutant for either red or recA, terminase stays bound to the left end of the open chromosome, inhibiting further transactions there (FEISS et al. 1983; KOBAYASHI et al. 1984; KUZMINOV et al. 1994). At the same time, the right end of the A chromosome is accessible to recombinational enzymes .and becomes a hotspot for recombination.

There are two ways to alleviate the dependence of the Red pathway on RecA to allow replication or to provide a second, non-allelic cut site with either cos or a restriction site. When replication is allowed, lambda recombination becomes relatively RecA-independent with exchanges no longer concentrated at the right end (STAHL et al. 1972a). It was proposed that lambda late DNA replication generates randomly distributed double-strand ends (SKALKA 1974; WILKINS and MISTRY 1974). Decreased recAdependence due to a restriction cut was demonstrated by STAHL et al. (1990). Data from a repetition of the restriction experiments are in Fig- ure 2.

Stimulation of Red+ R e d - recombination by replication or by restriction cutting could be taken to mean that the Red pathway can promote invasion in the absence of RecA if a double-strand break other than the one at cos is provided. The Red proteins could conceivably substitute for RecA in catalyzing invasion of these ends into intact duplex DNA. The relative inability of the Red machinery to work at cos in recA mutant cells could be explained if RecA protein is required to make the right end of linearized lambda accessible to Red enzymes, for example, by displacing bound terminase (KUZMINOV and STAHL 1997). However, this possiblility is weakened by the ability of an inverted cos site in the middle of the A chromosome to stimulate RecA-independent recombination in the absence of replication (STAHL et al. 1985; STAHL and STAHL 1985). Apparently, the recombination enzymes of A act at (one side of-) a cut cos as they do a t any other double-strand end.

The generation of non-allelic cuts could be releasing the Red pathway from RecA dependence by providing substrate for recombination by annealing (Figure 1, C- G) , When only standard cos is available as an initiation site, there is no opportunity to generate the requisite overlapping single strands. The crosses in Figure 2B support this hypothesis by demonstrating that in the absence of RecA the location of the restriction cut rela-

Genetic Recombination in Phage A 965

A

U n

c-

D y , b- F

E n U G -

FIGURE 1.-The two homology-dependent mechanisms for doublestrand end repair. (B and C ) The doublestrand end is processed by an exonuclease that creates a singlestrand overhang. (D and E) Invasion. (F and G) Annealing.

tive to the outside markers influences the ability to stimulate recombination. When the restriction cut is made on the Rts parent, there is little stimulation compared with that seen when the restriction cut is made on the Jts parent (compare Figure 2, B2 and B3). The asymmetry in the result reflects an asymmetry inherent in the cross. Recombinants were enumerated by selecting for the I? marker on the right arm of the Jts parent and thef marker on the left arm of the Rts parent. Anneal- ing from the right cos on the Jts parent to the restriction cut site on the Rts parent does not produce a scorable recombinant, while annealing from the restriction site on the Jts parent to cos on the Rts parent does do so. This interpretation is supported by the observation that the total yields of phage for the crosses in Figure 2, B2 and B3 are identical (recalling that dimerization by “recombination” is essentially prerequisite for packaging when DNA replication is blocked). Unscorable, double mutant recombinants would combine with a few “recombinants” between phage of identical genotype to produce equivalent total phage yields.

We note that all the results showing stimulation of Red recombination by doublestrand breaks in the absence of RecA can be explained by supposing that much of the recombination proceeds by annealing (Figure 1, C - G ) . Indeed, two non-allelic double-strand ends (facing in opposite directions, if they are polarized ends) appear to be provided in all these cases; one break is at the standard cos, the other is at a restriction site, or at an additional, inverted cos, or at hypothesized

randomly distributed ends of sigma-replication. Degra- dation of the 5“ending strands of these duplex ends by lambda exonuclease would expose complementary DNA sequences that could anneal with the help of lambda RedP protein, forming a recombinant duplex. RecA is not needed in such a reaction.

With only one double-strand break (at the regular cos), A recombination is largely RecAdependent, pro- ceeding by invasion. RecAdependence is appreciably relieved when a second double-strand break, provided at a location non-allelic to the first one, allows annealing. As described in the Introduction, the two possible mechanisms of A recombination differ not only in their RecAdependence, but also in the number and relative location of doublestrand breaks required and in the length of generated hDNA regions. RecAdependent invasion should happen whenever either one of the two parental DNAs is cut and should generate a short hDNA region marking the invasion of the end. In contrast, RecA-independent annealing should happen only when the two parental DNAs are cut at non-allelic locations and should generate a long hDNA region, spanning the entire length between the two cuts. To challenge our understanding of A recombination in the presence or absence of RecA, we tested the two predictions.

Physical analysis of annealing recombination: The two non-allelic double-strand breaks in the crosses above (Figure 2) differ in both efficiency and timing. Whereas a restriction cut is introduced into phage DNA right after injection with an efficiency of 90% (see below), cos-cutting starts 20 min later, reaching its peak by 40 min, when some 55% of cos sites are open (MYERS et al. 1995). To deliver synchronous double-strand breaks, we prevented cos-cutting altogether and cut both parents in vivo with the same restriction enzyme, but at non-allelic unique sites. Although the absence of co+ cutting makes genetic analysis in such a cross difficult (but not impossible: WILKINS and MISTRY 1974), recombining DNAs can be isolated from cells and analyzed directly.

The phages used in our analysis are diagrammed in Figure 3A. One parental phage bears the spi6 deletion eliminating the natural unique XhoI site, but cames a unique XhoI site -7 kb to the right of the deletion (SstII :: XhoI). The other parent retains the natural unique XhoI site opposite the spi6 deletion in the first phage, but bears the nin5 deletion, which almost over- laps the XhoI site in the first phage (Figure 3A). In addition, both phages are Pszls80 and have A immunity. Since the cross is carried out in Su- cells harboring a plasmid expressing A repressor, phage gene expression and DNA replication are severely inhibited. Thus, the particular genetic content of the phages is irrelevant, and we may think of the phage chromosomes as a pair of DNA substrates. The cells always harbor two compati- ble plasmids, one expressing a XhoI-isoschizomer re-


0

m ' 6 35

4

3

2

1

B1

?AA \

ce,,,

B2 J c 4

B3 J Vc

a - Density FIGURE 2.-Replication-blocked crosses to assess stimulation of recombination by a restriction cut delivered in vivo to one

parent. Both parents in each cross are density labeled, and the recovery of a single Heavy/Heavy peak of progeny phage attests to the adequacy of the block to DNA replication. Crosses ofjtsl5 c I 8 5 7 r X J cI' Rh2 are at restrictive temperature in dnaRts22 strains that carry the PmR7 restriction system on a plasmid (pPAORM3.8). This system is an isoschizomer of XhoI and cuts at A's solitary XhoI site, in the j-cI interval at 33.5 kb from the left end of A's 48.5-kb chromosome. The strains are Su- and the phages are Psw80, so that DNA replication is "double-blocked." Phages carry the int4 mutation to eliminate any involvement of A's site-specific Int system. In A, the host was the rec' strain FA77[pPAORM3.8]; in B, the host was FZ14[pPAORM3.8], a recA56 mutant version of that strain. Cross 1: both parents were uncuttable by virtue of being PmR7-modified. In cross 2, the R h parent was cuttable. In cross 3, the j t s cI parent was cuttable. JR+ recombinants were selected at 40" on the SuII' indicator K12SH-28, and plaques were scored as clear (~18.57) or turbid (cI') or mottled (cI/c' heteroduplexes). A, total phage; 0,s cI' R+ recombinants; 0, J cI R+ recombinants; 8, J d/c ' R+ recombinants. These crosses repeat those reported as Figures 2 and 3 in STAHL et al. (1 990).

striction endonuclease, PaeR7, the other expressing A ferred to a growth medium, and total DNA from the CI repressor and various combinations of A red and gum cells is purified at various times thereafter. DNA is cut functions. with restriction enzymes and fractionated by agarose

After adsorption of phages to cells on ice, the phages gel electrophoresis; subsequent blot hybridization la- are allowed to inject their DNA, infected cells are trans- bels the bands of interest. Uncut parental fragments,


cut parental fragments, and the two recombinant fragments are all separated (Figure 3, B and C).

We camed out the protocol with both rec' and recA mutant cells harboring four different plasmids: one that provides no A recombination functions, one that provides only Red functions, one that provides only Gam, and one that provides both Red and Gam (Figure 3, B and C). The following observations are common to both the ret+ and recA mutant cells. (1) Upon injection, phage DNA is cut by the restriction endonuclease, giv- ing two fragments of the expected size. (2) These fragments are degraded if Gam function is not supplied. (3) In the presence of both Gam and Red, the fragments are less stable than in the presence of Gam alone, suggesting that Reda catalyzes degradation (CJ: lanes h and k). (4) By 120 min after injection, recombinant bands are seen in the Red' crosses. ( 5 ) There is more recombinant product formed when both Red and Gam functions are supplied than when only Red is supplied. Since this latter difference is observed also in a recB mutant (not shown), it could reveal a recombination- stimulating effect of the inhibition of SbcCD nuclease by Gam (KULKARNI and STAHL 1989). A similar "stimulating" effect of Gam on Red-promoted recombination in the absence of RecBCD with these plasmids has previously been noticed (POTEETE and FENTON 1993). The main difference between the rec' and recA mutant cells is in the yield of the recombinant product, which is lower in the absence of RecA (by about the same factor observed in Figure 2, A3 us. B3). Minor differences include the following: (1) formation of recombinant product in rec' Gam cells, compared with its apparent absence in the case of recA mutant cells (cf : Figure 3, B and C, lanes i), in accord with genetic crosses showing that A recombination in rec" cells is only partly dependent on Red (SIGNER and WEIL 1968) even when DNA replication is blocked (STAHL et al. 1974); (2) relative stability of restricted DNA in recA mutant cells in the absence of recombination functions (CJ: Figure 3, B and C, lanes b), explained by the finding that some cells in a recA culture are defective in DNA degradation (KUZMI- NOV and STAHL 1997). We conclude that the dependence of recombinant band formation on both Red and Gam functions shows that this is a true recombinant formed by the Red pathway of phage A.

Further characterization of the recombinational reaction: In this physical assay, the phage chromosomes are simply substrates, since the recombinational enzymes are provided from plasmids. Therefore, there should be an optimal multiplicity of infection, at which the amount of the injected substrate DNA matches the recombinational capacity of the cells. We varied the multiplicity of infection of the parental phages and found that the best yield of the recombinant product is at- tained at a multiplicity of about five for each parental chromosome in both rec" and recA mutant cells (Figure

4, A and B). Points of secondary interest in these gels are as follows: (1) relative stability of restricted DNA at high multiplicity of infection (Figure 4, A and B, lanes a and c), suggesting titration of the Reda exonuclease activity; (2) appearance of irrelevant, plasmid-specific bands at the low multiplicity of infection (Figure 4, A and B, lanes f, h, j and 1). These bands were identified using DNA purified from cells before infection (not shown).

To establish the time course of the recombination reaction, we purified total DNA over time, quantifying relevant bands in the resulting blots by AMBIS (Figure 5 ) . The time course revealed the following. (1) In both rec' and recA mutant cells, cleavage of the incoming phage DNA with XhoI is completed by 20 min, with -90% of the input DNA being restricted. (2) The linearized DNA is gradually degraded in both backgrounds. (3) In both backgrounds the recombinant band first becomes detectable 20 min after injection (Figure 5 , A and C , lanes g), in accord with analogous observations of others (POTEETE and FENTON 1993) and also in (possibly fortuitous) agreement with the lag in recombinant DNA formation during a regular A infection (WILKINS and MISTRY 1974). (4) The accumulation of recombinant product starts to plateau -2 hr after injection. The relative quantity of the recombinant product reaches 3% of the input DNA in the rec" cells and 0.2% in recA mutant cells, in good agreement with the genetic results (Figure 2). Indeed, the best recombinant phage output in recA crosses was -lo6 phage particles, while the input was lo9 particles of each parental phage, putting the best recombinant yield at lo-' per infecting phage. We conclude that the formation of recombinant DNA by the Red pathway is slow in both rec" and recA mutant cells.

Two cuts are required for Red-mediated recombinant formation in the absence of Re& To test the notion that the mechanism of Red-promoted recombination in the absence of RecA is primarily by annealing rather than invasion, we compared the effect of cutting one substrate with that of cutting both. Invasion is possible if either one of the parental phages is cut, while annealing is possible only when the two parents are cut at non-allelic sites. To distinguish between these two possi- bilities, we constructed two additional phages, which are exactly like their spi6 or nin5 parents but lack their XhoI sites. Four possible pairwise combinations of the four phages included the original one in which both parents were cut, the two combinations in which only one of the parents was cut, and the one in which neither phage was cut (Figure 6). The cutting of only one parental phage in these crosses can be seen as the inequal- ity in uncut species (6 lanes i and 1 with f and 0) or as the production of only one cut species (CJ: lanes h and k with e and n).

The results of four such crosses in rec" cells (Figure


A 8.64 kb

Aspi6 parent

t cloned Xho I

13.6 kb

7.95 kb J

s p i 6 t natural XhoI

probe I I

Aspi6 Anin5 recombinant B

Time after 1 7 120 1 7 1 2 0 1 7 1 2 0 injection (min) r

Anin5

Red Gam 1 7 120

a b c d e f g h i

6A) demonstrate that the recombinant band forms whenever at least one of the parents is cut. We have compared the recombinant band formed in the “cut X cut” cross by matching intensities of its sequential dilutions with those from crosses where a single substrate DNA was cut (not shown). As a rule, the signals

j k I

FIGURE 3.-Recombi- nation promoted in repressed, restricted phage DNA by Red and Gam supplied from plasmids. (A) The scheme of the two substrate DNAs, the recombinant DNA, and the probe ( A fragment between bp 34823 and 36113) used to detect them. The spi6 and nin5 regions are boxed in the DNAs that retain them; boxes are absent in the DNAs having the corresponding deletions. The unique XhoI restriction sites for in uivocutting and the NcoI restriction sites used in the subsequent in vitro analysis are shown. (B) The rec’ experiment. Cells: 594; phages: MMS 2660 (Anin5) and MMS2663 (Aspib) . (C) The recA experiment. Cells: FS3809; phages: MMS2539 ( A n i n 5 ) and MMS2597 (Aspib) . The two substrate pairs give in- distinguishable results in both host genotypes. The supplied combinations of A recombination functions or their absence are shown as Red, Gam or- at the top. The molecular weight markers (all cut with NcoI) are as follows: uncut parents, MMS2661 and MMS2662; cut parents, MMS2660 and MMS2663; recombinant, MMS2666. The approxi- mate size of the fragments is as follows: Anin5 uncut, 13575 bp; Aspi6 uncut, 12500 bp; recombinant fragment, 9695 bp; Aspi6 cut, 8640 bp; Anin5 cut, 7945 bp.

from crosses in which only one parent is cut is 50% or greater than that from the cut X cut cross, suggesting that most of the recombinant product in the cut X cut cross is formed via invasion, although some may result from annealing. The same four crosses in recA mutant cells show that the recombinant band is formed only

Genetic Recombination in Phage A

C c E

a b c d e f g h i j k l m n o

when both parents are cut (Figure 6B). The dilution comparison revealed that in recA crosses the recombinant products from cut X cut crosses need to be diluted at least eightfold to be indistinguishible from the inde- tectable recombinant signal in the crosses where only one parent was cut (not shown). On the basis of this test we propose that Red-promoted recombination in the presence of RecA is mostly by invasion, whereas in its absence it is mostly by annealing.

A multiplicity of infection 30 20 10 6 3 1

nmanw uuuuuu lnjectlon(mln) 10 80 10 80 10 80 10 80 10 80 10 80

U m pCmW8 =f

recombln8nt + Cut pa- Q

a b c d e f g h i j k l

B multiplicity of infection 3 2 1 6 8 4 2 1

nmsthn HUUHUH lnjectlon(mln) 10 80 10 80 10 80 10 80 10 80 10 80

umpCm-S

w w - 3 Nconlbin8mt+

a b c d e f g h i j k l

FIGURE 4.-Optimal multiplicity of infection of the two parental phages. E. coli cells: rec', 594; recA, FS3809. Bacteria carried the Red Gam plasmid (pTP666) in addition to the PueR7 plasmid. The phages are MMS2539 (Anins) and MMS2597 (Aspic;). (A) ret+ cells. (B) recA mutant cells. The multiplicities of infection, expressed as a number of phage particles of each genotype per bacterium, are shown above the corresponding pairs of lanes. The desired multiplicities were obtained with varied cell concentration and constant phage concentration.

969

FIGVRE 3.-Con/inutd

In the absence of Red, recombinant phages are often hybrid all the way between the two doublestrand break sites: We tested genetically the prediction of long stretches of hDNA for Redcatalyzed recombination in the absence of RecA. Annealing predicts hDNA ex- tending from one doublestrand end to the other, while invasion envisions shorter hDNA, concentrated in the vicinity of a break site. The doublestrand breakstimu- lated crosses of STAHL et dl. (1990) suggested this possibility. When the Jts parent was cut, <lo% of the recombinants in the red+ cells were manifestly heteroduplex [forming mottled plaques, first described in T4 (HERSHEY and CHASE 1951)] for the middle marker, whereas in w d mutant cells, more than half of these recombinants were mottled (CJ: cross 3 in Figures 2 and 3 of STAHL et al. 1990). When the crosses were repeated here, the effect was comparable (CJ: Figure 2, A3 and B3).

To generalize the results of Figure 2B and to establish the properties of markers to be used in subsequent crosses, we performed replication-blocked R e d - crosses between phages with selectable markers near the ends of the h chromosome (with mutations in B or in R, respectively) and with a different CI allele (Figure 7A). As in the J X R crosses of Figure 2B, when neither parent is cut, the resulting recombinant yield is low, and most of the @R+ recombinants are cI+, indicating that they arose in the right interval, cI-R, close to cos. When the Bts R+ parent is cut at the XhoI site there is a 300-fold stimulation of recombination between Band R, and there are heteroduplex cI/cI+ phages, detected as mottled plaques, comprising about half of all the recombinants (Figure 7A).

In this cross, the marker that we use to score heteroduplex recombinants is located asymmetrically in the recombination interval, closer to the restriction cut than to cos. To verify that recombinants are hybrid not only at the left end of the interval but also at the right

970 M. M. Stahl et dl.

B

D

C f c

g c c a 5 E z n Time after injection (min) 2 0 0 0

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A f E g f cut cut uncut uncut

2 5 cut uncut cut uncut n e 5 X X X X

5 Z E o IO m o IO 70 o IO 70 o IO 70 nmsRor

L a b c d e t g h i J k l m n o

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injection (min)

a b c d e f g h i j k l m n o

FIGURE 6.-The four possible combinations of cut and uncut DNA substrates in recA and WT cells. rec’ cells: 594; r e d mutant cells: FS3809. A phages: Aspi6 cut, MMS2735; Aspi6 uncut, MMS2737; Anin5 cut, MMS2739; Anin5 uncut, MMS2740. Multiplicities were four of each phage type. (A) ret+ cells. (B) recA mutant cells. The four configurations of the cut and uncut substrates are shown above the gel pictures; in each pair, the top attribute refers to the Aspi6 phage, the bottom attribute refers to the Anin5 phage. The molecular weight markers are described in the legend to Figure 3. In this experiment we used A mutant phages to rule out the possibility of terminase production by repressed phages that might otherwise induce an unwanted double-strand break at cos.

end, we modified the cross to include an additional marker (Ssus7) to score heteroduplexes close to cos. The results of this cross, shown in Figure 7B, reveal that 90% of the particles selected for being B+ and biparental near cos (SF) are also heteroduplex at cI, near the restriction cut. When individual progeny plaques were picked off the Ssus7“permissive host, FS1585, at 32” (permissive for the ts markers) and spotted for the presence of 9 and of B+F, 60% (33/55) of the B ’ F progeny plaques spotted on the host non- permissive for Ssus7, demonstrating their biparental nature near cos. This estimate, like others of heteroduplexes, is a minimal one. These results support the view that most of the XhoI-stimulated recombinants are hybrid all the way from the XhoI site to cos.

In the two previous crosses, the restriction site is -4 kb from the CI marker used to score heteroduplex re-

combinants. To make sure that the high proportion of heteroduplex progeny was not due merely to the physical proximity of the restriction cut, we translocated the XhoI site -25% of the length of the chromosome to the left, increasing the distance between the cut site and CI to 16 kb. The new position of the restriction cut had only a modest effect on the overall result (compare Figures 7C and 2B3): (1) XhoIcutting increases the ab- solute yield offR+ recombinants, and (2) about half of the XhoI-stimulated recombinants are manifestly heteroduplex (mottled) for cI/c”.

We conclude that Redcatalyzed recombination in the absence of RecA generates recombinant phages that are hybrid all the way from cos to the restriction site, as predicted by the annealing mechanism.

We have interpreted the high heteroduplex frequency as resulting from annealing between complementary chains that owe their generation to doublechain cuts at the two non-allelic sites, XhoI and cos. In re& recA- replication-blocked crosses, the right end of A, generated by terminase cutting at cos, is accessible for genetic recombination, while the left end is blocked, presumably by terminase (FEISS et al. 1983; KOBAYASHI et al. 1984; KUZMINOV el al. 1994). Thus, for a XhoI site to stimulate heteroduplex formation at cI, cI must be located between the XhoI site and A’s right end. To test this prediction, we translocated the XhoI site to the right of the cI locus (Figure 7D). XhoI cutting at the new site (SslII :: XhoI) stimulates recombinant formation to the same extent as when the XhoI site is to the left of c1, but none of the detected recombinants is heteroduplex at cI. In fact, essentially all carry cI+, the allele from the XhoI-uncuttable parent, as expected. (Since we counted about 100 recombinants at each point, “essentially all” means, roughly, >99%, and “none” means el%.)

Distribution of exchanges in annealing recombma- tion: Because heteroduplexes can be underenumer- ated, the crosses reported in previous figures set lower limits on the fraction of cut-stimulated events that result in hDNA from the restriction site to cos. The c’s and c+’s of previous figures may be either misscored (or mismatchcorrected) heteroduplexes or recombinants that are not hybrid across the CI locus. To distinguish these alternatives, we conducted replication-blocked crosses in which only one parent contains heavy iso- topes. In such a replication-blocked “heavy-by-light” cross, the density of a given recombinant phage particle reflects the DNA contribution to that particle from each of the two parental phages (STAHL et al. 1972a, 1974).

FIGURE 5.-The time course of the recombination reaction. Bacteria as in Figure 4. The phages are MMS2539 (Anin5) and MMS2597 (Aspib) at a multiplicity of five. (A) Gel of the experiment in rec+ cells. (B) Graph based on four blots like one in A, quantified by AMBIS (a direct radioactivity scanner). a, uncut parental DNAs; 0, cut parental DNAs; crossed squares, recombinants. ( C ) Gel of the experiment in recA mutant cells. (D) Graph based on four blots like one in C , quantified by AMBIS. a, uncut parental DNAs; 0, cut parental DNAs; crossed squares, recombinants. The molecular weight markers are described in the legend to Figure 3.


5

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I I I I - Density FIGURE 7.-The production of heteroduplex recombinants by Red in the absence of R e d . Crosses are under the conditions

described in Figure 2B. (A) The results of Figure 2B are generalizable to crosses with other markers. In the cross Btsl cI26 X Rts129, cutting the Bts CI parent in vivoat position 33.5 kb increases the production of B+R+ recombinants, and most recombinants are heteroduplex at the cI locus. (Left) Both parents uncuttable (modified). (Right) The Bts cI26 parent was cuttable (unmodified). Phage were assayed on C600 at 32" for total titer and at 42" for B+R+ recombinants. A, total phage (where measured); 0, cI' R+ recombinants; 0, B+ CI R+ recombinants; 8, B' cI/c+ R+ recombinant$. (B) Many heteroduplexes extend from d , near the restriction cut, to S, near cos. The density label allows the separation of progeny phage (right peak) from unadsorbed phage (left peak). The cross was unmodified Btsl cI26 Ssw7 X modified Rts129. Phage were assayed on FS1585 at 32" for total titer (A) and at 42" for B+ R+ recombinants (A). Plaques on strain C600 at 42" select E'SF phages that are CI ( O ) , c+ (O), or mottled (8). (C) Heteroduplexes arise at CI even when the restriction site is moved far from cI. In both parents the standard XhoI site (at 33.5 kb from A's left end) has been inactivated and a new XhoI site has been inserted at 21.2 kb from the left end (see MATERIALS AND METHODS). In the left panel, both parents are uncuttable by being modified. In the right panel, theJts cI parent is cuttable at the cloned XhoI site. Phage were assayed on the SuII' host K12SH-28 at 32" for total titer and at 42" f o r r


If all of the XhoI-stimulated B+R' recombinants arise by annealing that extends from the XhoI site to cos, they will have a unique density. The predicted density when a cuttable light Bts parent is crossed with an uncuttable heavy Rts parent is that of a particle that has its entire chromosome from the heavy Rts parent except for a single light strand that runs from the XhoI site to cos. The XhoI site used for these experiments was present at the site of A's leftmost EcoRI site, 56% from the right end, so the resulting B+R' recombinants are expected to band at a position indicating that they are (100 - 56/2) = 72% heavy. If the clear and turbid recombinants seen in the previous Heavy X Heavy crosses are misscored (or mismatchcorrected) c/c" heteroduplexes, all recombinants will band at this density. If, on the other hand, there are substantia1 numbers of particles that are not hybrid over their entire length, the density distribution of recombinants will be broader (from nearly 100% to 44% heavy), and the CI alleles will be sorted according to density (Figure 8A).

The cross reveals that when the restriction site is far to the left of c1 -l/* of the B+R+ recombinants are mottled for cI, as in the cross of Figure 7C. However, the B+R+ recombinants do not fall into a single peak at 72% Heavy but, instead, sort out by density indicative of the three types. Clear (cI) recombinants form a peak at -47% density, implying that they arise by a rather simple break-join to the right of the cut XhoI site in the light, Bts cI857 R' parent. The turbid (cI+) recombinants form a fully heavy peak, like the one seen in the cross between uncuttable parents. The formation of these recombinants in such quantities is not predicted by the annealing model and will be addressed in DISCUSSION. Finally, the cI/cI+ heteroduplex phages do form a peak at the density expected for particles that are heavy on one chain and light on the other from the cloned XhoI site to the right end. The clear and turbid peaks both have shoulders at the density of the heteroduplexes, demonstrating the reality of misscored (or mismatch-corrected) heteroduplexes. Thus, the recombinants at the position expected for hDNA from the restriction site to cos (72% heavy) represent -64% of the total recombinant progeny, and the prediction of the annealing mechanism is, to a large extent, fulfilled.

DISCUSSION

We used replication-blocked crosses of phage A in which one or both parental phages were cut at unique

sites to distinguish between two mechanisms of recombination promoted by X's Red system. The imasion mechanism predicts that (1) the recombination is RecA-dependent, (2) only one of the two parents needs to be cut, (3) relatively short regions of hDNA are formed. As demonstrated by the present study, this mechanism is realized when recombination occurs in recA+ cells; it is the only mechanism available when only one of the two recombining molecules is cut. By contrast, A recombines poorly in recA- cells when only one parental phage is cut. This result is in agreement with the observation that the Red-catalyzed reaction, in which a cut A phage acquires genetic markers from an intact plasmid, requires RecA (CLW and BELFORT 1992). Others have examined recombination stimulated by an in vivo restriction cut to one parent in replication-blocked A crosses and also concluded that a solitary double-chain break was recombinagenic in a re& cross only when RecA was available (POTEETE and FEN-

In our crosses that examined intracellular phage DNA, we used the deletions spi6 and nin5 as markers. THALER et al. (1987) reported that heterology inter- fered with Red' R e d - recombination that was initiated by a restriction cut in vivo. If that interference was specific to invasion, the use of deletions as markers could contribute to our failure to see recombinant DNA arising in response to a restriction cut delivered to only one parent in our crosses.

When the two parental DNAs are cut at non-allelic locations, recombinant phage particles form in the absence of RecA (STAHL et al. 1985, 1990; STAHL and STAHL 1985). This recombination is demonstrably Red- dependent by a factor of lo4 (data not shown). We demonstrate that many of these Reddependent recombinant phages are hybrid over the entire length between the two cuts, implying that they were formed by annealing. This conclusion is substantiated by the results of restriction analysis, which show the formation of recombinant product when the Red recombination system operates in recA mutant cells on DNA substrates with overlapping ends. Thus the Red system of A, in the absence of RecA, catalyzes recombination primarily by annealing whereas, when R e d is available, Red promotes predominantly invasion recombination between a linear and a circular substrate.

Results less easily explained within the annealing model: The annealing model predicts that in density- labeled crosses there should be a single recombinant

TON 1993).

R+ recombinants. 0,s c" R+ recombinants; @,J CI K+ recombinants; @,f cI/c+ R+ recombinants. (D) There are no heteroduplexes at cI when the restriction site is moved to the right of cI. The standard XhoI site in both parents has been inactivated, and a XhoI site has been inserted into A's SstII site at 40.4 kb, to the right of cI8.57 (at 37.7 kb) (HAGEMANN and ROSENBERG 1991). In the left panel, both parents are modified; in the right panel, the Jts CI parent is cuttable. The progeny banded in a single H/H peak, which was assayed on the SUII+ host K12SH-28 at 32" for total phage and at 42" for SF recombinants. A, total phage; recombinants as in C.


I a, CI i= 0

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Density

Genetic Recombination in Phage X 975

species of a particular density, indicative of hDNA stretching from the restriction cut to cos. When we tested this prediction, we found three density species instead of one (Figure 8). The expected recombinant peak comprises -64% of all the recombinants, whereas the other two recombinant peaks signlfy exchanges close to either break site. This result argues that many of the recombinants scored as CI or c+ are not misscored cI/c+ heteroduplexes. Were they so, they would have banded with the heteroduplexes, as did some of both the CI and c+ plaques. These recombinants of distinctive density may have arisen by invasion, if such a reaction is possible in the absence of R e d (as we suspect it is, see below). On the other hand, these recombinants could conceivably have arisen by an act of annealing confined to the neighborhood of the cut site (illustrated for the CI recombinants in Figure 8C).

The interpretation of annealing as the primary route to recombinant formation in red+ recA- crosses is illustrated by Figure 2, B2 and B3, which shows that XhoI- cutting of theJs parent yields moreJR+ recombinants than results from cutting of the other parent. However, the production of fR+ recombinants is moderately stimulated by the cut in Rts parent, suggesting either that the left end of A is sometimes available for recombination in Red+ RecA- crosses or that Red acting in vivo can promote a low level of invasion (and see STAHL et al. 1990, Figure 3). The latter idea is supported by the observations that, in the absence of a provided restriction cut, Red can promote (a low level of) recombinants at A’s right end in R e d - cells (e.g., Figure 2B1 and the left panels of Figures 7, A, C and D, and 8B). STAHL et al. (1982) showed that proximity to cos determined the location of such recombinants. This activity may require A functions in addition to Red, since it is manifest in our “genetic” crosses but not in our “physical” crosses between repressed phages. The lack of detectable recombinant frequencies at the left end of linearized A presumably reflects the strong binding of terminase, the cos-cutting enzyme, at A’s left end (FEISS et al. 1983; KOBAYMHI et aZ. 1984; KUZMINOV et al. 1994).

DNA replication makes Red-mediated A recombination less Reddependent, suggesting that A DNA repli- cated in recA mutant cells is a good substrate for annealing. As described in the introduction of this paper, we have previously proposed (STAHL et al. 1990) that repli-

cation provides DNA ends throughout the genome, presumably as tips of rolling circle tails. Additional support for this interpretation can be garnered from reinter- preting an apparent paradox in STAHL et aZ. (1990). Those authors demonstrated that a restriction cut delivered to one of the two parents in a replication-blocked cross stimulates Red-independent recombination. However, features of the stimulation differed according to the method used to block chromosome replication. In that paper, and as illustrated in this one, when replication was blocked with mutations in the replicating machinery, the resulting RecA-independent recombination was predominantly by annealing, as shown by the dependence of yield on which parent was cut and by the high frequency of heteroduplex recombinant progeny. When replication was blocked by immunity in the 1990 paper, however, there was no evidence for annealing as a route to recombinant formation: cutting of either of the two imm’ parents was comparably effec- tive at stimulating recombination, and heteroduplexes were conspicuously scarce. We propose that the opera- tive difference between the two methods of blocking DNA replication is that one (the immunity block) is conducted in the presence of freely replicating hetero- immune “helper” phage, which are doubly mutant so that they cannot contribute to the formation of the selected recombinants. When a chromosome of one of the imm’ phages is cut by the restriction enzyme, it may execute invasion with a (probably supercoiled) circular A chromosome, producing by that route a scorable recombinant. A candidate for annealing with another immA phage, however, will have to wait until terminase provides cuts at cos. Before that, the replicating hetero- immune population will have provided a plethora of opportunities for annealing, which will divert the candidate into a nonproductive event.

The slowness of recombinant formation as revealed by our analysis of Red+ RecA’ crosses is harmonious with the observation by HILL et al. (1997) of the long- lived nature of single-stranded DNA recombination in- termediates in vivo. The lengthy interval between cutting and resection, on the one hand, and the appearance of completed recombinant DNA on the other is one of several striking similarities between Red+ RecA+ recombination and meiotic recombination of Sacchare myces cerevisiae

FIGURE 8.-Heavy X Light replication-blocked R e d - cross to assess the fraction of recombinants arising by annealing from a XhoI site to cos. (A) A scheme of the cross producing the recombinant product expected according to the annealing mechanism of Figure 1, C-G. (B) Light Btsl cI857was crossed by heavy Rts129. The crosses, in strain FZ14[pPAORM3.8], were Int- and double-blocked for DNA replication as previously. In the left panel, the Bts parent was uncuttable; in the right panel the Bts parent was cuttable. The progeny, separated by density, were plated on the SuIP strain C600 at 32” for total phage and at 42” for B+R+ recombinants. Symbols as in Figure 7A. The limits of the total phage distributions define the positions for progeny particles carrying fully heavy and fully light chromosomes, respectively. (C) A “partial annealing” scheme for the formation of the unexpected light, clear recombinants arising in the cross in B. In this scheme, the initial annealing step is not invariably followed by full strand assimilation. Instead, redundant, unpaired single strands may be clipped off. Alternatively (and probably) the unexpected recombinants arise by Red-mediated invasion effected at low level in the absence of RecA.


This work was supported by grant GM-33677 from the Institute for General Medicine of the National Institutes of Health and MCB- 9402695 from the National Science Foundation. F.W.S. is American Cancer Society Research Professor of Molecular Biology.

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CARTER, D. M., and C. M. RADDING, 1971 The role of exonuclease and p protein of phage A in genetic recombination. 11. Substrate specificity and the mode of action of A exonuclease. J. Biol. Chem. 246: 2502-2510.

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CASSUTO, E., T. LASH, IL C. SRIPRAKA~H and C. M. RADDING, 1971 Role of exonuclease and /? protein of phage A in genetic recombination. V. Recombination of A DNA in vitro. Proc. Natl. Acad. Sci. USA 6 8 1639-1643.

CLARK, A. J., and A. D. MARGULIES, 1965 Isolation and characterization of recombhationdeficient mutants of Escherichia coli K-12. Proc. Natl. Acad. Sci. USA 53: 451-459.

CLW, J., and M. BELFORT, 1992 Trans and cis requirements for intron mobility in a prokaryotic system. Genes Dev. 6: 1269- 1279.

ECHOLS, H., and R. GINGERY, 1968 Mutants of bacteriophage A defective in vegetative genetic recombination. J. Mol. Biol. 3 4 239- 249.

ENQUIST, L. W., and A. S a m , 1973 Replication of bacteriophage A DNA dependent on the function of host and viral genes. I. Interaction of red, gam, and rec. J. Mol. Biol. 7 5 185-212.

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Communicating editor: N. L. CRAIG

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Annealing Invasion in Phage A Recombination · ful repair of a double-strand end involves interac- tion with homologous DNA (RESNICK 1976, 1978; KRASIN and HUTCHINSON 1977). Such