Genetic Functions Promoting Homologous Recombination in Escherichia Coli: a Study of Inversions in Phage X

Genetic Functions Promoting Homologous Recombination in Escherichia coli: A Study of Inversions in Phage X

Don G. Ennis,*$'Susan K. Amundsen+**and Gerald R.

"Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, and TFred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104 Manuscript received June 19, 1986 Revised copy accepted September 13, 1986

ABSTRACT We have studied homologous recombination in a derivative of phage X containing two 1.4-kb repeats in inverted orientation. Inversion of the intervening 2.5-kb segment occurred efficiently by the Escherichia coli RecBC pathway but markedly less efficiently by the X Red pathway or the E. coli RecE or RecF pathways. Inversion by the RecBCD pathway was stimulated by Chi sites located to the right of the invertible segment; this stimulation decreased exponentially by a factor of about 2 for each 2.2 kb between the invertible segment and the Chi site. In addition to RecA protein and RecBCD enzyme, inversion by the RecBC pathway required single-stranded DNA binding protein, DNA gyrase, DNA polymerase I and DNA ligase. Inversion appeared to occur either intra- or intermolecularly. These results are discussed in the framework of a current molecular model for the RecBC pathway of homologous recombination.

0 elucidate the molecular mechanism of homol- 1974, 1975). KLECKNERand ROSS(1980) described a T ogous recombination it is important to identify derivative of phage X that has the potential to undergo the genetic functions that promote recombination. homologous intramolecular recombination. This The major pathway of recombination associated with phage contains three copies of ISIO, the terminal 1.4 conjugation and generalized transduction in Esche- kb of transposon TnlO (Figure 1). In addition to the richia coli is the RecBC pathway (CLARK1973). Pre- insertion in the rex gene of TnlO, with its two copies vious studies have shown that this pathway requires of ISIO, there is an insertion in or near the ral gene RecA protein (CLARKand MARGULIES 1965), Rec- of a third IS10 whose orientation is opposite to that BCD4 enzyme (EMMERSONand HOWARD-FLANDERS of the left ISIO element in the TnIO. Recombination 19677, and single-stranded DNA binding protein between these latter two ISIO elements can invert the (SSB) (GLASSBERG,MEYER and KORNBERG1979), and intervening 2.5-kb DNA segment, which contains the is stimulated by Chi sites (STAHLand STAHL1977). PL promoter of X (Figure 2). In one orientation of the RecA protein and SSB cooperatively promote the intervening segment transcription from PL activates exchange of DNA strands between double-stranded the red and gam genes; expression of one, or both, of DNA (dsDNA) and at least partially single-stranded these genes is necessary for plaque-formation on recA DNA (ssDNA) (reviewed by RADDING1982). RecBCD mutants of E. coEi (Fee+ phenotype) (ZISSLER, SIGNER enzyme unwinds and cleaves linear dsDNA (reviewed and SCHAEFFER1971a). In the other orientation nei- ther red nor gam is expressed, and the phage can form by TELANDER-MUSKAVITCHand LINN 1981). In this plaques on a P2 lysogen of E. coli (Spi- phenotype) paper we report additional functions required for the (ZISSLER, SIGNERand SCHAEFER197 lb). These phe- RecBC pathway of recombination. notypes allow the selection of phages with inversions With the eventual goal achieving homologous of from populations with the intervening segment ini- recombination in a cell-free system, we sought a re- tially in either orientation. KLECKNERand ROSS(1980) combination event that could occur within one DNA showed that inversion from the Spi- orientation to molecule. Such a monomolecular (intramolecular) re- the Fec+ orientation depends upon the host RecA action allowed X site-specific recombination to be de- function and is stimulated by the host RecBCD func- tected in cell-free extracts for the first time (NASH tion; these results showed that the inversion in this phage occurs by homologous recombination. ' Present address: Department of Molecular and Cellular Biology, Uni- versity of Arizona, Tucson, Arizona 85721. In this paper we examine the roles of other host * Present address: Department of Urology, Northwestern University Med- functions and of Chi sites in inversion of derivatives ical School, 303 East Chicago Avenue, Chicago, Illinois 6061 I. ' To whom correspondence should be addressed. of this phage. With suitably marked derivatives under ' The RecBCD enzyme or exonuclease V, formerly designated RecBC appropriate infection conditions we examine the abil- enzyme, has recently been shown to contain three subunits, coded by the re&, recC and reCD genes (AMUNDSENet al. 1986). ity of the inversion to occur intramolecularly or inter-

Genetics 115: 11-24 (January, 1987) 12 D. G. Ennis, S. K. Amundsen and G. R. Smith

,"434 N PL Tn IO Fec+ c -c e-- 1 c- .-.c c. ,de1267e 3, I 2 3 FIGURE1 .-Abbreviated map of phage A. The orientations of the three copies of IS10 (shaded boxes) in A366 (KLECKNERand It Ross 1980) are indicated by the arrows under the boxes. Also Tn 10 shown are the locations of genes A, J. red, gam, N, cl, and R: Spi- deletions b221, de1267, and de1317: Chi sites x+A, x+B, x+C, and c -t c. x+D; and the imm'" substitution. The distances between the mark- 1 2 3 ers are not drawn to scale. FIGURE2.--Inversion of A366 between the Fec+ orientation and the Spi- orientation (from KLECKNERand Ross 1980). The top line molecularly. We discuss the role of each of the iden- shows the structure of the Fec' orientation, in which transcription tified functions within the framework of a molecular (wavy line) from the PLpromoter proceeds leftward across the gam model of recombination proposed for the RecBC gene, whose expression is required, in the red-3 background, for growth on E. coli RecA- mutants but whose expression blocks pathway (SMITHet al. 1981). growth on a P2 lysogen (ZISSLER,SIGNER and SCHAEFER1971a; LINDAHLet al., 1970). Recombination between the left pair of IS10 MATERIALS AND METHODS elements (shaded boxes) inverts the N-PL segment to produce the Spi- orientation (bottom line), in which PL-promotedtranscription Bacterial and phage strains: These are listed in Tables does not activate gum, with the resultant opposite growth pheno- I and 2, respectively, with their genotypes and sources. types. Strains V419 [lig-7(ts)], S883 [gyrB203(ts)], V265 [ssb-l(ts)], V268 [dnaB39l(ts)], V270 [dnaM(ts)], and V278 Mol- the frequency of inverted phage following infection A48U(ts)] were confirmed to be unable to form colonies at 41 '. Strains V268 and V270 were complemented for high of test bacteria in which one or another recombina- temperature growth by lysogenization with a XdnaB+ trans- tional pathway was operative (Table 3). These path- ducing phage from C. GEORGOPOULOS(data not shown). ways have been described by CLARK(1 973). Inversion Strain S883 (gyrB221(couR))was resistant to 25 Fg of cou- was most frequent in wild-type bacteria (such as strain mermycin per milliliter. The Feb- phenotype of strains S877 594) in which recombination proceeds principally by and V418 was confirmed by their failure to support the growth of Xred-3 (ZISSLER, SIGNERand SCHAEFER1971a). the RecBC pathway. After a single cycle of infection

The ability of strain V278 to grow at 41 O was restored by with phage 1388 (TnlU Chi+) the frequency of in- transduction with phage 1104 (XpolA); similarly the Feb+ verted phage was 64 (k28) X low4,while the fre- phenotype was restored to strain S877 by lysogenization quency in the input phage was less than 0.2 X with this phage. Inversion was much less frequent by the RecE or RecF Determination of inversion frequency: Host bacteria growing exponentially in TB + maltose (1 % Difco Tryptone, pathways: the frequencies were 4 X 10-4 and 1 X 10-4 0.5% NaCl, 1 Pg of thiamine/ml, 0.1% maltose) were in- in strains JC86'79 and JC9387, respectively, although fected at about 2 X IO* cells/ml with phage; after 15 min a control experiment involving coinfection with phage adsorption, the mixture was diluted 1: 100 into warm TB + 1388 and Xspi-8 cI857 S7(am) showed that the RecE maltose and gently shaken at the indicated temperature for and RecF pathways were as proficient as the RecBC 1.5-4 hr. CHCIRwas added to kill residual bacteria. Titers of total phage and Fec+ phage were determined as indicated pathway in producing interchromosomal exchange in the tables. recombinants (data not shown). To test inversion by Inversion was studied using phage X strain 1388, desig- the X Red pathway, a Ximm4j4 Red+ helper phage was nated A366 by KLECKNERand ROSS (1980). Phage 1388 used to coinfect a RecA- RecB- host (strain S796), bears the red-3 mutation to inactivate the A Red pathway of and the progeny were differentially titered on appro- recombination and contains one or more Chi sites in the TnlO insertion. Some studies reported here used derivatives priate lysogens. Inversion by the Red pathway was of phage 1388 bearing a deletion of part of TnlU removing infrequent: Red+ helper produced an inversion fre- this Chi site, but bearing mutations creating a functional quency of 3 X lo-", while the control Red- helper Chi sequence at other known locations in A. Stocks of these produced no detectable increase in inversion fre- phages in the Spi- orientation were prepared by growth in quency over that in the input. RecA- RecB- bacteria (strain S794 or S796) to limit the production of phage in the Fec+ orientation. Following a In strains AFT162, AFT163 and AFT181, the single cycle of infection in test bacteria, the frequency of RecE and RecF pathways are expected to have little inversion was measured by titration on RecA- hosts (for if any activity, since they do not contain the recE gene, Fec+ inversion phage) and on RecA+ hosts (for total phage). they are sbcB+, and they are recF143. Thus, recombi- For convenience, inversion frequencies are expressed in nation in these strains is expected to be principally by units of IO-4. the RecBC pathway, as the data in Table 3 show. RESULTS Inversion was frequent in the RecBCD+ strains, but in recB21 or recC73 isogenic strains no significant Recombinational pathway specificity of inver- increase in inversion frequency over that of the input sion: We first tested the ability of several recombina- phage was observed. tional pathways to support inversion by determining A class of mutants designated RecBCD* is pheno- Genetic Functions for Recombination 13

TABLE 1 E. coli K-12strains

Strain Alternate designatioil designation" Genotype Source or reference

594 lac-3350 galK2 galT22 rpsLl79 WEICLE(1 966) C600 thr-I leuB6 thi-1 EacYl supE44 tonA21 APPLEYARD(1954) JC8679 thr-1 leuB6 ara-14 ProA2 lacy1 tsx-33 galK2 his-4 rpsL3I xyl-5 GILLEN(1974) mil-J argE3 thi-l recB2J recC22 sbcA23 supE44 JC9387 As JC8679 but sbcA+ sbcB15 sup' GILLEN(1 974) QR48 thi recA supE44 SIGNERand WEIL (1968) S60 1 trp gal recA SMITH(1975) s794 478 recA56 recB21 supE gal endA F. STAHL S796 recA1 recB21 gyrA (Hfr) SCHULTZ,TAYLOR and SMITH (1983) S87 1 809 As 594 plus (XJ13(ts) int-4 red-3 gam-210 R5(am)) M. STAHL S876 TO43 gal malB rpsL I. HERSKOWITZ S877 TO44 gal malB rpsL polA I I. HERSKOWITZ S882 N99 rpsL galK2 M. GELLERT S883 N4177 galK2 rpsL gyrB203(ts)gyrB221(couR) MENZELand GELLERT(1983) AFT162 thr-1 leuB6 ara-14 proA2 lacy1 tsx-33 galK2 his-4 rpsL31 91-5 SCHULTZ,TAYLOR and SMITH mtl-1 argE3 thi-1 recFl43 rac- (1983) AFT1 63b As AFT162 plus recB21 SCHULTZ,TAYLOR and SMITH (1983) AFT181 As AFT162 plus recC73 SCHULTZ,TAYLOR and SMITH (1983) V66 his-4 met rpsL31 galKZ xyl-5 argA21 recF143 rac- SCHULTZ,TAYLOR and SMITH (1983) v74 As V66 plus recC343 SCHULTZ,TAYLOR and SMITH (1983) v75 As V66 plus recB344 SCHULTZ,TAYLOR and SMITH (1983) V218 As V66 plus recD1009 argA+ CHAUDHURYand SMITH(1 984) V219 As V66 plus reCC1010 argA+ CHAUDHURYand SMITH(1 984) V265 KLC436; thyA36 rha-8 malA7 amp-50 deoC2 ssb-J(ts) VALES,CHASE and MURPHY(1980) CGSC6525 V266 KLC434; As V265 but ssb+ VALES,CHASE and MURPHY(1 980) CGSC6526 V267 As C600 plus thyA R. SCLAFANI V268 E391 As V267 plus dnaB39l(ts) R. SCLAFANI V270 PC6 thyA leu rpsL dnaB6(ts) R. SCLAFANI V278 CGSC5642 trpA33 polA480(ts) (=polA exl) KONRADand LEHMAN(1 974) V302 DM2558 srlC300::TnIO A(Eac-gpt)5 rpsL ENNISet al. (1985) V303 DE192 A(lac-gpt)5 rpsL lexA5I(Def) sulA2Il ENNISet al. (1985) V304 DE406 As V303 plus malB::Tn9 DM49, malB::Tn9 X DE192c V305 DE407 As V304 plus lexA3(Ind-) ENNISet al. (1985) V306 DE274 As V303 plus recA730 srlC300::TnIO ENNISet al. (1985) V417 N 1624; relA1 sPoTI bglR6 rpsL50 X- GOT-TESMAN,HICKS and GELLERT CGSC5066 (1973) V418 N1626 AS V417 PIUS lig-4 GOTTESMAN, HICKS and GELLERT CGSC5068 (1973) V419 N2668; As V418 plus lig-7(ts) GOTTESMAN,HICKS and GELLERT CGSC5069 (1973)

a Strain designations in the cited references, if different from our collection designation. CGSC strains were provided by B. BACHMANN from the E. coli Genetics Stock Center. This strain was incorrectly listed as AFT162 by SCHULTZ,TAYLOR and SMITH(1983). ' Constructed by P1-mediated transducted with a malB::Tn9 derivative of strain DM49 as donor and strain DE192 as recipient.

typically recombination proficient but exonuclease V- is independent of recE and recF (CHAUDHURYand deficient (CHAUDHURYand SMITH 1984). Some of SMITH 1984). The data in Table 3 show that the these mutations are in recD, coding for a third subunit pathway operating in RecBCDt mutants was profi- of RecBCD enzyme, and one (recCIOIO) is in recC, cient for inversion, and that in the absence of Chi it whose product presumably interacts with that of recD was about ten times more proficient than the wild- (AMUNDSENet al. 1986). The recombination profi- type RecBC pathway. (Compare strain V66 with ciency of RecBCD* mutants depends upon recA+ but strains V218 and V219.) Inversion in the RecBCD* 14 D. G. Ennis, S. K. Amundsen and G. R. Smith TABLE 2 and STAHL1985). The following experiments deter- Phage X strains mined a role for Chi sites in stimulating inversion and the dependence of the frequency of inversion on the Strain position of Chi relative to the invertible segment. The designa- rion Genotype" Sourceb or reference de1267 and del317 deletions remove most of the unique sequence of TnlO (Ross, SWANand KLECKNER N7(am) N53(am) Our collection 834 1979) (Figure 1). These deletions also remove the Chi 858 x+A1?1 N7(am) N5?(am) Our collection 965 red-3gam-210 xfC151 Our collection site(s) in TnlO, as shown by a comparison of plaque 980 "(am) N53(am) x+C157 Our collection morphology of Red- Gam- TnlO del and Red- Gam- 1028 x+B121 imm" cl(ts) Our collection TnlO phages on ret+ bacteria: the former phages 1079 j442(am) Our collection formed small plaques, while the latter formed large 1104 ppolA c1857 nin-5 Q73(am) KELLEY,CHALMERS and S7(am) MURRAY(1977) via S. plaques under the same conditions, characteristic of ROGERS Chi+ mutants (HENDERSONand WEIL 1975). Into the 1383 J6(am) zmm434R5(am) M. STAHL del derivatives we introduced x+ mutations at the XC 1384 j6(am) red-3 imm434 M. STAHL and XDloci to the right of the invertible segment and RS(am) at the XAand XB loci to its left (STAHL,CRASEMANN 1385 b145? imm" cl(ts) M. STAHL 029(am) x+D123 and STAHL1975). Since Chi stimulates recombination 1386 622 1 rex-173: :Tn 10 ROSS, SWANand KLECK- primarily to its left (STAHLet al. 1980), the x+C and de1267 e1857 NER (1979) x+D mutations, but not the xfA and x+B mutations, 1387 b22 1 rex-l7?::Tn I0 Ross, SWAN and KLECK- would be expected to stimulate inversion. del31 7 ~1857 NER (1979) As shown in Table 3, inversion in recBCD+ hosts 1388 6221 red-3 raE(?)::ISlO KLECKNERand ROSS rex-173::TnIO ~1857 (1980) (strain X366) occurred about 20 times more frequently in phage 1389 As 1388 but Tn'lO de1267 1388 X 1386 1390 (x'C) than in phage 1389 (x'). The stimulation 1390 As 1389 plus x+C157 1389 X 980 was observed only when the test bacteria were 1391 As 1389 plus ~'Al31 1389 X 858 recBCD+. This result is consistent with previous obser- 1389 X 1385 1392 As 1389 plus x+D123 vations that Chi stimulates interchromosomal ex- 1400 red-? gam-210 imm434 Our collection x'C157 change only by the RecBC pathway (GILLENand 1412 As 1389 plus x*B12I 1389 X 1028 CLARK1974; STAHLand STAHL1977; CHAUDHURY 1413 As 1388 but TnlO de1317 1388 X 1387 and SMITH1984). The degree of stimulation of inver- 1416 As 1413 plus x+D123 1413 X 1385 sion depended upon the distance between the Chi site 1418 As 1390 plus N7(am) 1390 X 834 N53(am) and the invertible segment: stimulation in strain 594 1421 As 1413 plus x'C157 1413 X 980 (recBCD+)decreased exponentially with distance by a 1424 As 1389 PIUS R5(dm) 1391 X 1384 factor of 2 every 2.2 kb (Figure 3). Stimulation by a 1426 As 1390 plusJ442(am) 1390 X 1079 - Chi site about 2 kb from the nearer end of the a See Figure 1 for the locations of genes, deletions and insertions. invertible segment was about 20-fold over the back- X, recombination during lytic growth of the indicated phages. ground level observed in a xo phage. Stimulation was hosts was not stimulated by Chi. Similar results have detectable with a Chi site as far as 10 kb away. x+A been found for biparental A vegetative crosses in these and x+B, Chi sites located to the left of the invertible strains (CHAUDHURYand SMITH1984). segment relative to the conventional X map, did not Inversion was not detectable in the RecA- RecB- stimulate inversion. In fact, the frequencies of inver- strain S796. Since phage 1388 in the Spi- orientation sion in phage 1391 (x+A) and phage 1412 (x+B)in grows poorly in RecA- RecBCD' bacteria (ZISSLER, strain 594 were fourfold lower than that in phage SIGNERand SCHAEFER197 la), we did not directly test 1389 (x'); this factor is similar to that by which a Chi+ the requirement for RecA. KLECKNER and ROSS site increases the burst size of XRed-Gam- (HENDER- (1 980) reported reduced inversion frequency of phage SON and WEIL 1975; MALONEand CHATTORAJ1975) 1388 in stocks prepared on mutant hosts: a tenfold and may reflect an enhancement, by these Chi sites, decrease on a recB2I mutant and a >60-fold decrease of total phage yield but not of Fec+ inversion phage. on a recAI recB2l host. Such a result would be expected if intracellular "mix- These observations show that inversion occurred ing" of phage chromosomes were not perfect (see most frequently by the RecBC pathway and markedly DISCUSSION) and if the x+A and x+B sites on phage less frequently by the RecE, RecF or Red pathway. chromosomes that had inverted could not stiniulate An explanation for this difference will be offered in formation of packageable dimers. the DISCUSSION. Certain mutations, designated TexA, in recB and Stimulation of inversion by Chi sites: Chi sites recC reduce, but do not abolish, stimulation of recom- stimulate the frequency of interchromosomal recombination by Chi in biparental X vegetative crosses bination in E. coli and phage X (reviewed by SMITH (SCHULTZ,TAYLOR and SMITH1983; LUNDBLADet al. Genetic Functions for Recombination 15

TABLE 3 Dependence of recombinational inversion frequency on recombinational pathways and Chi sites

E. coli host for infection” Frequency (X lo4)of inversion of phage 1388 1389 1390 Strain rec genotype Rec pathway (TnlO Chi+) X0 x+c 594 rec+ Mostly RecBC 64 (9) 12 (9) 201 (11) JC8679 recB.21 recC22 sbcA23 RecE 4.0 (4) 7.6 (3) 8.0 (2) JC9387 recB.21 recC2.2 sbcBl5 RecF 1.0 (4) 5.6 (3) 4.7 (1) S796 recAl recB21 “None” 0.18 (5) 1.9 (4) 4.0 (1) S796 recA1 recB.21 Redb 2.9 (1) ND‘ ND AFT162 A recEd recF143 RecBC 29 (3) 9.8 (1) ND AFT163 A recE recF143 recB.21 “None” 0.30 (1) 0.79 (1) ND AFT181 A recE recF143 recc73 “None” 0.29 (1) 1.1 (1) 2.1 (1) V66 A recE recF143 RecBC 16 (2) 4.3 (4) 92 (4) V218 A recE recF143 recD1009 RecBCDS ND 54 (3) 48 (3) V219 A recE recF143 recc1010 RecBCDS ND 33 (3) 34 (3) Frequency of Fec+ phage in input 0.20 0.82 0.84 The indicated host strains were infected with the indicated invertible phage at an m.0.i. of 0.1 (except as noted in footnote b), diluted, and incubated for 2 hr as described in MATERIALS AND METHODS. Fec+ inversion phage were titered on strain S601 (RecA-) and total phage on strain JC8679 (RecBC- SbcB-). The data reported are the means of the frequencies determined from the number of infections indicated in arentheses. Standard derivations ranged from 0.23 to 0.44 times the mean. ‘These cells were coinfected with a Red+ helper phage (1383) at m.0.i. = 5.0. Titrations of the invertible phage were done on Ximm434 lysogens. Coinfection with an isogenic red-3 helper phage (1384) produced a Fec+ inversion frequency of 0.16 X 1O-4. ‘ ND, not determined. A recE means rac- (i.e., deleted for the cryptic rac prophage in which the recE gene resides).

region, we estimate maximal inversion frequencies of about 350, 80 and 40 (X10-4) in ret+, recB344 and recC343 cells. These values are in reasonable agree- VIOOk\021 ment with Chi activities, measured in biparental X 0 vegetative crosses by the method of STAHLand STAHL (1977), of 5.8, 2.4 and 1.6 in these cells, respectively (SCHULTZ,TAYLOR and SMITH 1983), or 6.1,2.0 and 1.8 (LUNDBLADet al. 1984). (By this method a value of unity indicates no Chi activity.) Consistent with these results is the markedly reduced Chi-dependent DNA cleavage activity in extracts of recC343 cells (PONTICELLIet al. 1985). Collectively, these results indicate that the RecBCD enzymes in TexA mutants have reduced ability to interact with Chi. As in rec+ cells, the stimulation by Chi in the TexA mutants decreases approximately exponentially with distance

024681012 between Chi and the invertible region. Thus, the Distance (in kbl from Chi Site defect in the TexA mutants may be primarily the to Invertible Seqment reduced interaction with Chi. FIGURE3.-Dependence of stimulation of inversion frequency In summary, inversion occurred most frequently by upon distance between a Chi site and the invertible segment. Data the Chi-stimulated RecBC pathway. The following are the averages of 3 to 11 single-cycle infections of strain 594 (ret+), V75 (recB344) and V74 (recC343). The phages were 1390, experiments used Chi+ inversion phages in RecBCD+ 1421, 1392, and 1416 (in order of increasing distance from the Chi bacteria. site to the invertible segment). The Chi-stimulated inversion fre- Requirement for recombination-promotingfunc- quency is the observed average frequency of inversion with each tions of E. coli: In order to determine if other E. coli phage minus the average frequency of inversion of the xo phage 1389. The x” frequencies were 12, 8.9 and 8.4 (X10-4) for the functions were required for inversion, hosts mutant three strains 594, V75 and V74, respectively. for (or blocked by an inhibitor of) one of several DNA metabolism functions were infected with the Chi-con- 1984). Chi’s stimulation of inversion was also reduced taining inversion phage. Comparisons were made in in cells with either of two TexA mutations, recB344 temperature-sensitive mutants at high and low tem- and recC343 (Figure 3). By extrapolating the curves peratures, in strains isogenic except for a mutation to zero distance between the Chi site and the invertible affecting a single function, or in the presence or 16 D. G. Ennis, S. K. Amundsen and G. R. Smith

TABLE 4 Dependence of recombinational inversion frequency on E. coli DNA metabolism functions

~ ~~~~ E. coli host for infection Frequency (X 104) of inversion phage" Burst sizeb Strain Relevant genotype Temperature Expt. 1 Expt. 2 Expt. 1 Expt. 2 V417 1ig+ 32 120 178 29 42 V417 1ig+ 41 85 153 33 38 V418 lig-4 32 110 182 6.9 8.9 V418 lig-4 41 0.24 0.06 0.4 0.8 V419 lig-7(ts) 32 7.3 1.3 0.1 0.6 V419 lig-7(ts) 41 0.18 0.07 0.03 0.05 V266 ssb+ 32 190 134 9.7 21 V266 ssb+ 41 285 154 20 28 V265 ssb- I (ts) 32 114 62 12 6 V265 ssb-1 (tS) 41 12.5 2.5 2 2 S876 polA+ 32 83 92 18 25 S877 polAl 32 1.4 12.3 3.6 2.6 V278 polA480(ts exo) 32 110 150 12 10 V278 polA480(ts exo) 41 87 101 9 8 V267 dnaB+ 30 98 22 V267 dneB+ 42 112 22 V268 dnaB39 I (ts) 30 197 120 10 10 V268 dnaB39l(ts) 42 177 79 0.8 1.4 V270 dnaBb(ts) 30 43.4 9.9 V270 dnaB6(ts) 42 41.6 0.3 "The indicated host strains were infected with phage 1390 (x'C) at m.0.i. of 0.08 to 0.25 and grown for 2-4 hr at the indicated temperature; inversion frequency was determined as the titer of phage on strain S601 (RecA-) divided by that on strain JC8679 (RecBC- SbcA-). In infections of dnaB strains, the infected cells were centrifuged and washed (twice) in warm TB + maltose medium to remove unadsorbed phage. Total progeny phage per infected cell. absence of an inhibitor. cation was blocked by the mutations except for pol- The results in Table 4 show that DNA ligase, single- A480. These results suggest that extensive DNA rep- stranded DNA binding protein, and DNA polymerase lication, mediated in part by DnaB, is not required I were required for high frequency inversion. Defects for inversion, but that local (repair) DNA synthesis, in any one of these functions reduced the frequency mediated in part by DNA polymerase I and DNA of inversion 1O-50-fold. The requirement for DNA ligase, is required. ligase was shown using strains carrying either the Eig- The role of DNA gyrase was first tested in strain 4 or the lig-7(ts) mutation (GOTTESMAN,HICKS and S883 carrying the gyrB203(ts) mutation (MENZELand GELLERT1973). The reduced level of functional DNA GELLERT1983). Inversion occurred at approximately ligase in the lig-7 strain at both the permissive and the same frequency at the permissive and nonpermis- nonpermissive temperature (GOTTESMAN,HICKS and sive temperature (Table 5). However, coumermycin GELLERT1973) was reflected by a low frequency of and nalidixic acid, inhibitors of the gyrase B and inversion in both cases. The requirement for DNA gyrase A subunits, respectively (GELLERTet al. 1976, polymerase I was shown using a strain carrying the 1977), reduced the frequency of inversion by 85-95% PolAI mutation, which is deficient in the DNA polym- in the gyr+ strain S882. Nalidixic acid, but not cou- erase activity (DE LUCIAand CAIRNS1969). The 5'- mermycin, inhibited inversion in the coumermycin- 3' exonuclease activity of this enzyme was not re- resistant strain S883. This result indicates that cou- quired, however, since a mutation @olA480) render- mermycin inhibited inversion via DNA gyrase and ing this activity temperature sensitive (KONRADand suggests that DNA gyrase is required for recombina- LEHMAN1974) did not significantly reduce the fre- tional inversion. [We presume that the residual DNA quency of inversion. Similarly, DnaB function did not gyrase activity in the gyrB203 (ts) mutant at 41" is appear to be required for inversion as shown by the sufficient to promote inversion.] similar frequencies of inversion at high and low tem- We hypothesized that coumermycin might inhibit peratures in strains with the dnaB39l(ts) mutation inversion due to induction of one or more SOS func- (WECHSLERand GROSS1971) or the dnaBb(ts) mutations controlled by the LexA repressor (LITTLEand tion (CARL1970). The reduction of phage burst size MOUNT 1982). This hypothesis is not supported by under restrictive conditions showed that the restric- the data in Table 5. The frequency of inversion was tive conditions were effective, as extensive multipli- not greatly altered whether the host strain contained Genetic Functions for Recombination 17 TABLE 5 Dependence of recombinational inversion frequency on E. coli DNA gyrase

Frequency (X 10') of inversion E. coli host for infection phageb Burst size' Temperature Strain Relevant genotype ("C) Drug Expt. 1 Expt. 2 Expt. 1 Expt. 2 S882 ar+ 37 None 352 254 40.7 30.8 S882 #r+ 37 cou 48.8 11.5 0.7 0.5 S882 or+ 37 Nal C6.9 11.1 0.5 0.2 S883 gyrB(ts, CouR) 32 None 133 156 2.7 3.5 S883 gyrB(ts, CouR) 32 cou 167 138 2.7 2.9 S883 gyrB(ts, CouR) 32 Nal <6.4 11.2 0.25 0.25 S883 gyrB(ts, CouR) 41 None 95 105 0.3 0.4 S883 gyrB(ts, CouR) 41 cou 38.4 21.7 0.2 0.3 S883 gyrB(ts, CouR) 41 Nal 18.2 15.8 0.2 0.3 V302 lexA+ 37 None 42 1 268 32 51 V302 lexA+ 37 cou 11.2 10 0.1 0.2 V303 lexA5 I( Def) 37 None 151 86 57 59 V303 lexA5 I (De0 37 cou 7.2 3.1 0.2 0.2 V304 lexA51(Def) 37 None 160 144 28 29 V304 lexA5 1 (Deg 37 cou 17 1.6 1.o 0.5 V305 lexA3(Ind-) 37 None 69 55 3.4 16 V305 lexA3(Ind-) 37 cou 3.4 1.3 0.6 0.8 V306 lexASI(Def) recA 730 37 None ND 202 ND 11.0 V306 lexA5 1 (De0 recA 730 37 cou ND 1.5 ND 0.8 Bacteria were infected 10 min after resuspension in TB plus 25 gg/ml coumermycin (Cou) or 25 rg/ml nalidixic acid (Nal). *The indicated host strains were infected with phage 1390 (x'C) at m.0.i. of 0.08 to 0.25 and grown for 2-4 hr at the indicated temperature; inversion frequency., was determined as the titer of phage on strain S601 (RecA-) divided by that on strain JC8679 (RecBC- Sbck-). ' Total progeny phage per infected cell.

1 a defective LexA repressor, and hence constitutive AJ c for SOS functions (strains V303 and V304), or con- c tained a noninducible LexA repressor (strain V305), or contained a RecA protein in the constitutively active state (strain V306). In each case coumermycin 3 inhibited inversion to about the same extent as in the lexAf strain V302. Collectively, the results in Table 5 3 2 indicate that DNA gyrase is required for recombinational inversion and suggest that this requirement is Intromolecular Intermolecular direct, presumably due to DNA supercoiling of the FIGURE4.-Schematic representation of intramolecular and intermolecular recombination. On the left a single reciprocal ex- inversion substrate or product. change between two IS10 elements (hatched boxes) in the same Intra- vs. intermolecular inversion: Inversion molecule inverts the intervening segment. On the right two non- could occur by either intra- or intermolecular inter- reciprocal exchanges between IS10 elements on different molecules actions (Figure 4). We used three approaches to de- invert the intervening segment. Other recombination events (not shown) between IS10 elements would produce additions and dele- termine which of these mechanisms prevails. The tions, some of which would not be viable. Only regions represented results suggest that both routes can lead to inversion. by hatched boxes with parallel arrows are in homologous alignment. In the first approach, cells were infected with fewer than one phage per cell (on the average) under con- either by rolling circle replication or by recombina- ditions blocking replication; a high frequency of in- tion; but recombination, of course, is not possible if version would imply that inversion can occur with just there is only one phage chromosome in the cell. one invertible chromosome per cell and thus can occur We therefore blocked replication by another intramolecularly. We noted in the preceding section means-repression by a homoimmune prophage that infection of dnaB(ts) E. coli mutants under restric- (THOMASand BERTANI1964) in the test bacteria- tive conditions resulted in inversion frequencies and allowed phage maturation to occur by recombi- nearly as high as those under permissive conditions nation with a heteroimmune helper phage incapable (Table 4). Since multimeric A-DNA is required for of inverting on its own or by interaction with the formation of mature particles (STAHL et al. 1972), phage 1388 derivative. The results in Table 6 show replication is presumably not entirely abolished under that inversion occurred at nearly as high a frequency these conditions, however. Multimeric A can arise during infection of the repressing lysogen (average of 18 D. G. Ennis, S. K. Amundsen and G. R. Smith

TABLE 6 Recombinational inversion in homoimmune infection

Phage* m.0.i. Burst sin? Frequency (X IO4) of inversion phaged Bacteria infected" Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. I Expt. 2 1390 0.07 0.03 0.8 0.7 108 92 s871 { 1400 9.5 2.1 37 38 0.09 0.03 1390 0.06 0.03 1.9 9.3 180 101 594 { 1400 9.1 2.1 44 58 0.02 0.08 S87 1 1400 9.5 2.1 70 46 0.007 0.01 594 1390 0.06 0.03 12.8 20.5 258 134.9

a Strain S871 is a h lysogen of strain 594. After the adsorption period, the infected cells were centrifuged and resuspended (twice) in warm TB + maltose medium to remove unadsorbed phage. Phage 1390 is x+C invertible phage; 1400 is red-3 gum-210 2mm4" helper phage. Full genotypes are in Table 2. ' Number of progeny phage of the indicated type per cell infected with that type. Fee' phage were titered on strain S601 (RecA-). For the mixed infections about 100 plaques were tested for immunity by toothpicking to lawns of appropriate lysogens. The frequency of inversion for each phage type was calculated from the total Fec+ titer times the fraction of its immunity type among the tested plaques. In each of the four determinations the fraction of immA(invertible) type ranged from 0.26 to 0.34.

100 x 10-4 from two experiments) as during infection TABLE 7 of the non-lysogen (140 X That the prophage Inversion of x+ and xo phage during coinfection" blocked replication of the invertible phage is indicated by the lower burst size in the lysogen (0.8 progeny Frequency (5 1 04) of inversion phage per cell infected with phage 1390) than in the phage non-lysogen (1.9 or 9.3 progeny phage per cell, de- X+ X0 pending upon the multiplicity of infection (m.0.i.) of Expt. x+ Parent' xo Parentb Phage Phage the helper phage). Although the repressed invertible ~~ 1 1390 (x'C) 1424 (xoR) 90 20 phage could have been passively replicated by the 1 1390 (x'C) 100 helper phage after recombination with it, the low 1 1424 (xoR) 6.2 burst size of the invertible phage indicates that such 2 1426 (x'CJ) 1389 (x') 190 22 replication, if it occurred, was not extensive. The 2 1418 (x+CNN) 1389 (xo) 210 29 2 1426 (x'CJ) 150 conclusion from these experiments agrees with that 2 1418 (X'C NN) 160 from the preceding section: inversion can occur with 2 1389 (x") 9.6 limited replication and therefore may occur intramo- In experiment 1, strain C600 was infected with an m.0.i. of 2.5 lecularly. of each phage type for mixed infections or 5.0 for single infections. In the second approach, cells were coinfected with In experiment 2 the m.0.i. was 7 for each phage. Growth was for a frequently inverting phage (containing x'C) and a 1.5 hr at 37". In experiment 1, inversion frequencies were calculated as follows: (1) Sus+x+ inversion frequency = titer of Chi' rarely inverting phage (containing x"). Since the stim- plaque-forming units (p.f.u.) on strain S601 (RecA-) f titer of large ulation by the Chi site is not expected to act in trans p.f.u. on strain 594(P2). Plaques on strain S601 were transferred with toothpicks to lawns of strain C600, on which the phage could except by recombinational interaction, a high fre- invert to the Spi- orientation. These phage were streaked on lawns quency of inversion of the xo phage would imply that of strain C600(P2) to score Chi+ (large) plaques. (2) Sus-x'inversion the x+ phage helps the xo phage by recombining with frequency = titer of Sus- Chi" p.f.u. on strain QR48 (SuII+ RecA-) ititer of small Sus- p.f.u. on strain C600(P2). Plaques on strain it, and thus that inversion can occur intermolecularly. QR48 were transferred with toothpicks to lawns of strain C600 The results in Table 7 show that the frequency of (SuII+) and strain 594 to score Sus- plaques. From the lawn of inversion of the phage was increased about three- strain C600 phage were streaked on lawns of strain C600(P2) to x'' score Chi' plaques. Small plaques on strain C600(P2) were trans- fold in three coinfection experiments; in each case the ferred with toothpicks to lawns of strain C600 (SuII') and strain frequency of inversion of the x+ phage, however, was 594 to score Sus- plaques. In experiment 2, inversion frequencies 15-20-fold higher than that of the singly infecting were calculated as follows: (1) Sus+ xo inversion frequency = titer x" of turbid p.f.u. on strain S601 (RecA-) + titer of small p.f.u. on phage and was about the same whether it infected strain 594(P2). (2) Sus- x+ inversion frequency = [titer of clear alone or with the xo phage. If inversion occurs solely p.f.u. on strain QR48 (SuII+ RecA-) - titer of clear p.f.u. on strain by the intermolecular route diagrammed in Figure 4, S601 (RecA-)] + [titer of large p.f.u. on strain C600(P2) (SuII+) - titer of large p.f.u. on strain 594(P2)]. In each case the subtrahend, the frequency of inversion of the xo phage should be representing recombinants between x+C and the sus marker, was one-half that of the x+ phage (provided intracellular 10% or less than the minuend; hence, the inversion frequencies would not be significantly changed if the corrections were not "mixing" of the phage DNAs is perfect). Since the xo introduced. frequency was only about 10-25% of the x+ fre- Full genotypes are given in Table 2. Genetic Functions for Recombination 19 TABLE 8 Outside marker exchange and recombinational inversion

~ FecC frequency (X 10') % Am+ among total phage % AmCamong Fecf phage

Infecting phage" Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 (x+C 150 120 4.4 4.0 16 12 14261418 (x'C J,NN) I 1426 (X'CJ) 180 94

a Complete phage genotypes are in Table 2. Strain C600 was infected at an m.0.i. of 7 of each phage. Growth was for 1.5 hr at 37°C. Mixed infections with phages 1426 and 141 8, and their single infection controls were assayed as follows: total Fec+ and Sus+ Fec+ p.f.u. were titered on strains QR48 (SuII' RecA-) and S601 (RecA-), respectively. Total phage and total Sus+ phage were titered on strains C600 (SuII+) and 594 (Su-), respectively. Other infections were assayed as described in TaMe 7 (experiment 2). ' Data from Table 7 (experiment 2).

quency, it appears from these results that inversion the frequency of inversion did not depend upon the can occur, perhaps primarily, by intramolecular re- input m.0.i.: infection of strain C600 with phage 1390 combination. On the other hand, since the frequency (x'C) at m.0.i. values of 11, 4.6, 2.4, 0.24 and 0.026 of inversion of the xo phage was increased (modestly) yielded inversion frequencies of 100, 120, 95, 110 by the presence of the x+ phage, some inversion events and 89 X respectively. may occur intermolecularly. The frequency of Fec' phage in the stocks used for The third approach was similar to the second, ex- infection varied slightly (Table 3). This variation did cept that the coinfecting phages each carried a differ- not detectably influence the frequency of Fec+ phage ent marker to the left (J),to the right (xC),or within in the progeny, however. When Fec+ stocks and Fec- (NN)the invertible segment. A higher frequency of stocks were mixed to produce input Fee' frequencies exchange of these markers among inverted phages of 0.17, 0.45, 0.72, 1.5, 2.0 and 5.2 x the than among total phages would imply intermolecular frequencies of Fec+ phage in the progeny from a inversion. In each of three cases examined, the ex- single-cycle infection of strain 594 (Rec') were 80, 93, change of the markers was greater among inverted 78, 200, 100 and 120 X respectively; input (Fec+) phages than among total phages (Table 8). In frequencies of 0.17, 1.5 and 5.2 X into strain the first case (1426 X 1418), one marker was to the S796 (RecA- RecB-) produced output frequencies of left of the invertible segment and the other marker 0.34, 2.5 and 13 X respectively. These results was in the middle; in the second case (1389 X 1426), indicate that the measured Fec+ frequency in the one marker was to the left and the other to the right; progeny of Rec+ infections largely reflects recombi- and in the third case (1389 X 1418), one marker was nation in the infected cells and not preferential repli- in the middle and the other to the right. In those cases cation or maturation of the Fec+ phage. involving a middle marker (NN),intermolecular in- The frequency of inversion as determined here version as diagrammed in Figure 4 would lead to could be inflated for the following reason. Upon in- 100% exchange of the markers, one-half of which version the phage expresses gam, whose product in- would be the type scored. Since the observed frequen- hibits the host RecBCD nuclease (UNGERand CLARK cies (1 4-25%) were intermediate between the theo- 1972); Gam-mediated inhibition of RecBCD nuclease retical 50% and the 34%in the total phage popula- allows X replication to switch to the rolling circle mode tion, it appears from these results that inversion may (ENQUISTand SKALKA1973). Consequently, the in- occur by a mixture of the intramolecular and inter- verted phage might be preferentially replicated by the molecular routes. rolling circle mode, thereby augmenting its frequency Additional observations on inversion; control ex- in the population. Such preferential replication would periments: The experiments reported above used result in cell-to-cell variation in the frequency of in- variable m.0.i. by the invertible phage. We found that version, since the average number of inverted prog- 20 D. G. Ennis, S. K. Amundsen and G. R. Smith eny per infected cell was small (about 0.5) (Table 4). We did not find significant cell-to-cell variation in infections of strain C600 with phage 1390 (x’C). An infected culture (m.0.i. = 0.1) of strain C600 was diluted and distributed to produce nine small cultures each with an average of 130 infected cells and another nine cultures with an average of 14. The frequency of inversion ranged from 110 to 200 (X10-4) for the first nine cultures and from about 100 to about 300 (x1 0-4) for the second nine, except for one culture in -- each set which had a frequency about twice that of -+ or + the others. (The small numbers of inverted progeny - I make these numbers somewhat uncertain.) The lack of “jackpots” suggests that replication “bonanzas” do FIGURE5.-Model of recombination promoted by RecBCD enzyme and Chi sites (modified from SMITHet al. 1981). See text for not make a major contribution to the observed fre- details. The open box represents RecBCD enzyme, which moves quency of inversion. from right to left on the first parental duplex DNA (pair of thin In a second approach we compared the average lines). The second parental duplex DNA (pair of thick lines) may burst size of inverted (Fec+) phage with the number be circular (see DISCUSSION) or may be connected to the first parental of Fec+ infective centers. The average Fec+ burst size DNA (for intramolecular recombination as diagrammed in Figure was 0.81 Fec+ phage per infected cell, and the fre- 4). quency of Fec+ infective centers, determined by plat- lyzed by DNA gyrase (GELLERTet al. 1976, 1977). ing the infected culture on a RecA- host (strain S601) Since the formation of stable hybrid DNA by purified immediately after infection, was 0.52 Fec+ infective RecA protein and SSB protein requires, in the absence centers per infected cell. The similarity of these num- of DNA topoisomerase I, a DNA terminus within the bers indicates that most of the cells produced about region of homology, we suppose that the DNA tail one Fec+ inversion phage and that very few produced produced by RecBCD enzyme in the invertible phage “jackpots,” in agreement with the previous experi- studied here must be degraded from the Chi site to ment. the homologous region. At some time before comple- DISCUSSION tion of the recombination event, restoration of the degraded DNA would require local (repair) DNA The results presented here show that inversion be- synthesis, likely by DNA polymerase I. Pairing pro- tween repeated sequences in X occurred at high fre- moted by RecA protein and SSB protein of the dis- quency when a Chi site was located to the right of the placed strand of the D-loop with the gap in the region repeated sequences and close to them, and when RecBCD enzyme, RecA protein, single-stranded DNA of homology produces a symmetric structure, the binding (SSB) protein, DNA ligase, DNA gyrase and HOLLIDAY(1964) junction (H). Cutting and reassocia- DNA polymerase 1 were present. tion of the previously uncut DNA strands produces a We interpret the functional requirements for inver- reciprocal exchange (I). DNA ligase is required to seal sion by the RecBC pathway in the context of the the nicks left here, at the earlier stage of cutting, and model for recombination shown in Figure 5 (SMITHet at the termination of DNA synthesis. al. 1981). In this model RecBCD enzyme binds to a The requirements for RecBCD-promoted inversion duplex DNA end (step A) (TAYLORand SMITH1985) reported here are consistent with earlier observations and moves along the DNA, unwinding and rewinding on the RecBC pathway acting on vegetative A. Mc- it, producing ever-growing single-stranded loops (B MILIN,STAHL and STAHL(1974) demonstrated that and C) (TAYLORand SMITH 1980). When RecBCD RecBCD-promoted interchromosomal recombination enzyme approaches a Chi site from the proper side of X’proceeds in the absence of replication. LAMet al. (from the right in the cases studied here), it cuts one (1974) and MCMILIN,STAHL and STAHL(1 974) found DNA strand about five nucleotides to the 3‘-side of that Chi-stimulation occurs in the absence of replica- Chi (D) (PONTICELLIet al. 1985; TAYLORet al. 1985). tion. On the other hand SIEGEL(1974) observed a Continued unwinding produces a single-stranded small amount of DNA synthesis associated with the DNA tail extending from Chi toward the “down- site of Rec-promoted exchange in X. DNA polymerase stream” direction (E). In a reaction promoted by RecA I and DNA ligase are likely enzymes involved in this protein and SSB protein (F) (reviewed by RADDING synthesis. These observations are consistent with the 1982) this DNA tail invades a homologous DNA du- observations reported here: inversion requires DNA plex to form a D-loop (G). D-loop formation occurs polymerase I and DNA ligase, but not DnaB function, most rapidly with a supercoiled recipient duplex (SHI- which is required for X replication (reviewed by BATA et aZ. 1979); intracellular supercoiling is cata- FURTHand WICKNER1983); in addition, inversion Genetic Functions for Recombination 21 occurs when replication is blocked in a homoimmune intermolecular inversion requires at least two phage infection. [HAYSand BOEHMER(1978) found that chromosomes per cell, inversion by this route would inhibitors of DNA gyrase reduce the frequency of be impossible in cells containing only one phage chro- recombination between direct repeats in UV-treated mosome. But since there is no guarantee that repli- repressed A, but this recombination is recB-indepen- cation is totally blocked, no firm conclusion can be dent.] drawn. Other types of RecBCD-promoted recombination The following evidence suggests intermolecular in- require RecA protein and SSB protein and are stim- version. First, coinfection of a Chif inversion phage ulated by Chi sites. A requirement for RecA protein increased the frequency of inversion of a Chi’ phage was demonstrated for X (Red-) interchromosomal re- (Table 7). Since Chi is a site (STAHL,CRASEMANN and combination by SIGNERand WEIL (1968) and for STAHL1975), such “helping” of the Chi’ phage by the conjugation- and transduction-associated E. coli re- Chi+ phage suggests that their two DNAs interact, combination by CLARKand MARGULIES(1965). A presumably by recombination. Second, exchange of requirement for SSB protein was demonstrated for E. markers flanking or within the invertible region was coli recombination by GLASSBERG,MEYER and KORN- greater among inverted progeny than among total BERG (1979). Stimulation by Chi sites has been dem- progeny (Table 8). The statistical coincidence of the onstrated for X interchromosomal recombination by marker exchange and the inversion suggests that they LAMet al. (1974) and MCMILIN,STAHL and STAHL occur in the same event (i.e., intermolecular inver- (1 974) and for conjugation- and transduction-associ- sion). The extents of “helping” and of marker ex- ated E. coli recombination by DOWERand STAHL change were not as great as would have been expected (1 98 1). The common requirements for these types of if all inversions were intermolecular and if intracellu- recombination suggest that they proceed by closely lar “mixing” of phage DNAs were perfect. Thus, while related mechanisms. the evidence rather strongly suggests some intermo- We noted that the degree of Chi stimulation is an lecular inversion, it fails to demonstrate that this is exponentially decreasing function of the distance be- the sole route. However, since a co-efficient of coin- tween the Chi site and the recombining chromosomal cidence greater than unity is observed for exchange segment (Figure 3); this observation suggests that of distant X markers (e.g.,AMATI and MESELSON1965), some event blocking Chi stimulation has a constant intracellular “mixing” does not seem to be perfect. probability of occurring per unit distance. From the Thus, inversion might be entirely by the intermolec- data in Figure 3 we calculate that the blocking event ular route but, nevertheless, produce the observed occurs once per 2.2 kb with a probability of 0.5. limited degrees of “helping” and marker exchange. Within the context of the model of recombination Recombination between direct repeats in X, to add shown in Figure 5, this event might be the failure of or delete one or more repeats, appears to occur by RecBCD enzyme to continue unwinding the DNA; the RecBC pathway principally by an intermolecular for example, RecBCD enzyme might have a constant mechanism (BELLETT,BUSSE and BALDWIN1971). probability of falling off the DNA rather than contin- These authors observed that in single-cycle infections uing to unwind. Alternatively, the blocking event phage with a duplication produced equal yields of might be the destruction of a recombinational inter- single-copy and triple-copy progeny phage. Whether mediate before DNA polymerase I and DNA ligase inversion occurs by intramolecular or intermolecular complete repair of the gap extending from the in- recombination cannot be determined by an analogous verted segment to the Chi site. The available evidence method. does not allow a choice between these or other possi- The requirement for DNA gyrase for inversion bilities. (Table 5) might be taken as evidence for intermolec- We attempted to determine whether the inversion ular inversion, given that RecBCD enzyme requires a events studied here occurred primarily by the intra- double-stranded DNA end for entry (TAYLORand molecular mechanism or by the intermolecular mech- SMITH1985) and that DNA gyrase requires circular anism diagrammed in Figure 4. We believe that the DNA to introduce supercoils (GELLERT1981). How- available evidence suggests that inversion can occur ever, both RecBCD enzyme and DNA gyrase might, by both routes, but no firm conclusion appears war- in the cell, act on the same DNA molecule: the DNA ranted from this evidence. might be recircularized after RecBCD enzyme enters, The following evidence suggests intramolecular in- as appears to be the case for X recombination (KOBAY- version. Infection at low m.0.i. (<0.1 phage per cell) ASHI et al., 1984) or DNA gyrase might act on linear under conditions inhibiting DNA replication (by a DNA, as appears to be the case for phage T7 DNA dnaB mutation or by a repressing prophage) yielded replication (ITOH and TOMIZAWA1977). Thus, inversion frequencies nearly as high as those from RecBCD enzyme and DNA gyrase could conceivably infections allowing replication (Tables 4 and 6). Since promote intramolecular inversion. 22 D. G. Ennis, S. K. Amundsen and G. R. Smith An additional line of evidence is consistent with the research was supported by research grants GM 32194 and GM view that inversion occurs principally by the intra- 31693 from the National Institutes of Health. S.K.A. was supported by training grant 5 T32 CA 09229, and G.R.S. by Research Career molecular mechanism. This mechanism requires that Development Award AI 00547, from the National Institutes of the recombination event be reciprocal (Figure 4). Health. There is a strong correlation between the ability of a recombination pathway to promote inversion and the LITERATURE CITED pathway’s apparent reciprocality. The RecBC pathway was more proficient in promoting inversion than AMATI,P. and M. MESELSON,1965 Localized negative interfer- ence in bacteriophage A. Genetics 51: 369-379. was the Red, RecE or RecF pathways (Table 3). The AMUNDSEN,S. K., A. F. TAYLOR,A. M. CHAUDHURYand G. R. “Kec” pathway, probably primarily the RecBC path- SMITH,1986 red: the gene for an essential third subunit of way, promotes principally reciprocal exhange (HER- exonuclease V. Proc. Natl. Acad. Sci. USA 83: 5558-5562. MAN 1965; MESELSON 1967; SARTHYand MESELSON APPLEYARD,R. W., 1954 Segregation of new lysogenic types 19761, while the Red pathway promotes principally during growth of a double lysogenic strain derived from Esch- erichia coli K12. Genetics 39: 440-452. nonreciprocal exchange in X vegetative crosses (WEIL BELLETT, A. J. D., H. G. BUSSE and R. L. BALDWIN,1971 1969; SARTHYand MESELSON 1976); similarities be- Tandem genetic duplications in a derivative of phage lambda. tween the genetic origins and functions of the Red In: The Bacteriophage Lambda, Edited by A. D. HERSHEY,pp, and RecE pathways are consistent with the view that 501-513, Cold Spring Harbor Laboratory, Cold Spring Har- bor, New York. RecE pathway may also be principally nonreciprocal CARL,P. L., 1970 Escherichia coli mutants with temperature sen- (for review see SMITH 1983). The low frequency of sitive synthesis of DNA. Mol. Gen. Genet. 109: 107-122. inversion by the Red, RecE and RecF pathways can CHAUDHURY,A. M. and G. R. SMITH,1984 A new class of thus be accounted for by the assumption that inversion Escherichia coli recBC mutants: implications for the role of is primarily intramolecular. RecBC enzyme in homologous recombination. Proc. Natl. Acad. Sci. USA 81: 7850-7854. X The frequent inversion in vegetative reported CLARK,A. J., 1973 Recombination deficient mutants of E. coli here contrasts with the infrequent inversion in the and other bacteria. Annu. Rev. Genet. 7: 67-86. bacterial chromosome (HILL and HARNISH198 1; CLARK,A. J. and A. MARGULIES,1965 Isolation and characteri- SCHMIDand KOTH 1983). We can account for this zation of recombination-deficient mutants of Escherichia coli difference if, as just argued, inversion requires recip- K12. Proc. Natl. Acad. Sci. USA 53: 451-459. DE LUCIA,P. and J. CAIRNS,1969 Isolation of an E. coli strain rocal recombination, promoted by the RecBC path- with a mutation affecting DNA polymerase. Nature 224 1164- way. RecBCD enzyme requires a double-strand DNA 1166. end to initiate DNA unwinding (TAYLORand SMITH DOWER,N. and F. W. STAHL,198 1 x activity during transduction- 1985). Such an entry site is provided at high frequency associated recombination. Proc. Natl. Acad. Sci. USA 78: at cos during packaging of vegetative X (KOBAYASHIet 7033-7037. EMMERSON,P. T. and P. HOWARD-FLANDERS,1967 Cotrans- al. 1982). A similar double-strand break may occur duction with thy of a gene required for genetic recombination only infrequently in the bacterial chromosome and in Escherichia coli. J. Bacteriol. 93: 1729-1731. hence allow only infrequent inversion. Other factors, ENNIS, D. G., B. FISHER, S. EDMISTONand D. W. MOUNT, however, may be responsible for the different fre- 1985 Dual role for Escherichia coli RecA protein in SOS quencies of inversion; for example, the inverted re- mutagenesis. Proc. Natl. Acad. Sci. USA 82: 3325-3329. ENQUIST,L. and A. SKALKA,1973 Replication of bacteriophage X peats in X studied here are about 2.5 kb apart, while DNA dependent on the function of host and viral genes. I. most of those in the bacterial chromosome are about Interaction of red, gam, and rec. J. Mol. Biol. 75: 185-21 2. 1O3 kb apart. FURTH,M. E. and S. H. WICKNER,1983 Lambda DNA replication. The recombination event studied here may be well pp. 145-173. In: Lambda II, Edited by R. W. HENDRIX,J. W. suited for an in vitro analysis of recombination. The ROBERTS,F. W. STAHLand R. A. WEISBERG.Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. possibility that inversion occurs intramolecularly GELLERT,M., 198 1 DNA topoisomerases. Annu. Rev. Biochem. might increase the frequency of this recombination 50: 879-910. event, relative to bimolecular recombination events, GELLERT,M., K. MIZUUCHI,M. H. O’DEA,T. ITOH and J. TOMI- in dilute DNA solutions. The availability in purified ZAWA, 1977 Nalidixic acid resistance: a second genetic char- form of the identified proteins required for inver- acter involved in DNA gyrase activity. Proc. Natl. Acad. Sci. USA 74: 4772-4776. sion-RecBCD enzyme, RecA protein, SSB protein, GELLERT, M., M. H. O’DEA, T. ITOH and J. TOMIZAWA, DNA gyrase, DNA polymerase I and DNA ligase- 1976 Novobiocin and coumermycin inhibit DNA supercoil- makes feasible an attempt to detect recombination ing catalyzed by DNA gyrase. Proc. Natl. Acad. Sci. USA 73: promoted by these purified proteins. 4474-4478. GILLEN,J. R., 1974 The RecE pathway of genetic recombination We are grateful to NANCYKLECKNER, FRANK and MARYSTAHL, in Escherichia coli. Ph.D. Thesis, University of California, IRAHERSKOWITZ, MARTIN GELLERT, BARBARA BACHMANN, STEVE Berkeley, California. ROGERS,BOB SCLAFANIand COSTAGEORGOPOULOS for phage and GILLEN,J. R. and A. J. CLARK,1974 The RecE pathway of bacteria. We thank GARYBRAEDT, FRED PONTICELLI, ELISSA SENA bacterial recombination. p. 123- 136. In: Mechanisms in Recom- and ANDREWTAYLOR for helpful comments on the manuscript and bination, Edited by R. F. GRELL.Plenum Press, New York. KAYSHIOZAKI for her skill and patience in its preparation. This GLASSBERG,J., R. R. MEYERand A. KORNBERG,1979 Mutant Genetic Functions for Recombination 23

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