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

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Genetic Functions Promoting Homologous Recombination in Escherichia Coli: a Study of Inversions in Phage X Copyright 0 1987 by the Genetics Society of America 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 intermolec- ularly. 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).
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