X Sequence Protects Against Recbcd Degradation of DNA in Vivo (Reca/Plasmid Replication/Recombination/Bacteriophage A/Rolling Circle Replication) PATRICK DABERT, S

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 12073-12077, December 1992 Genetics

X sequence protects against RecBCD degradation of DNA in vivo (RecA/plasmid replication/recombination/bacteriophage A/rolling circle replication) PATRICK DABERT, S. DUSKO EHRLICH, AND ALEXANDRA GRUSS* Laboratoire de G~ndtique Microbienne, Institut de Biotechnologie, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France Communicated by Frank Stahl, September 24, 1992

ABSTRACT RecBCD is a multifunctional enzyme in- cies that may correspond to nonterminated replication inter- volved in DNA degradation and homologous recombination. It mediates (17, 18). In E. coli as well as in other organisms, we also produces an endonucleolytic cleavage near properly ori- observed that insertion of foreign DNA into rcr replicons ented X sites (5'-GCTGGTGG-3'). Plasmids are not known to often provokes a shift in plasmid profile from monomeric to be affected by either RecBCD enzyme or the presence of a X predominantly high molecular weight linear form (HMW) (17, site. We report here that plasmids that replicate by a rolling 18). HMW consists of tandem head-to-tail plasmid direct circle mechanism accumulate large amounts of high molecular repeats. Such molecules should be a substrate for RecBCD weight linear multimers (HMW), either if they contain a X site exonuclease (Exo). We proposed that the foreign DNA or if RecBCD is absent. An in vivo inducible system for rolling inserts, mainly G+C-rich, served to block degradation of the circle replication was constructed to study RecBCD and its long-tailed o form, presumably by affecting RecBCD (18). interactions with X. Results show that (1i) MW accumulation Here, we report that insertion of a X sequence in a rcr is X orientation dependent, and (ii) a succession of X sites plasmid, or inactivation of RecBCD, results in HMW accu- prevents degradation of HMW by RecBCD enzyme. These mulation. Furthermore, we show that a succession ofX sites results demonstrate X activity in plasmids. The rolling circle can protect HMW from degradation by RecBCD in vivo, mechanism produces a a structure during plasmid replication; possibly by titrating active RecBCD. Studies in recA mutants we propose that the double-stranded DNA tail of this ar form lead us to propose that, in our system, RecA serves to protect allows RecBCD entry; the tail is degraded unless it is protected the ends of linear DNA from access by RecBCD. by a X site. By analogy, a principal role of X in the survival of A redgam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degrada- MATERIALS AND METHODS tion by RecBCD. Strains and Phage. Recombination proficient E. coli strains are HVC45 (leu-6 thi-J pro lacYl tonA supE44 hsdR str; The RecBCD enzyme has principal roles in both DNA laboratory collection), JM101 [A(lac pro) thi supE F'traD36 degradation and homologous recombination in Escherichia proAB 1acJqZ AM15; ref. 19], and AB1157 (thr-1 leu-6 proA2 coli (1, 2). The enzyme is multifunctional; it acts as a his4 thi-J argE3 lacYl galK2 ara-14 xyl-S mtd-i tsx-33 single-stranded (ss) and double-stranded (ds) DNA exonu- supE44 rpsL31 kdgKSJ; ref. 18). JM101 was the host strain clease, helicase, ATPase (see refs. 3 and 4 for review), and used for all induction experiments. Recombination defective site-specific endonuclease which recognizes the 8-base-pair strains, derived from AB1157, are recA13 (AB2463 provided (bp) sequence, 5'-GCTGGTGG-3', known as X (5). RecBCD by R. Devoret, Centre National de la Recherche Scienti- recognizes and nicks 3' ofX ifit enters a dsDNA end from the fique, Gif sur Yvette, France), recB21 recC22 (JC5519 pro- right of the X site as written here (6). It is proposed that a 3' vided by A. Cohen, Hebrew University, Israel), and recA13 tail formed after X nicking by RecBCD stimulates homolo- recB21 recC22 (JC5547 provided by C. Dohet, Institut gous recombination to the left of X (4, 6-8). Jacques Monod, France). Bacteriophage T4 gene 2am mutant The in vivo interaction of RecBCD with X and its effect on (T4 2-), kindly provided by W. Wackernagel (University of recombination were elucidated by using bacteriophage A as a Oldenburg, F.R.G.), was used to monitor RecBCD Exo model system (9-12). In this paper, another system is de- activity; the protocol is described in the legend of Fig. 4. scribed to study RecBCD-X interaction. We are studying Medium, Bacterial Transformation, and DNA Preparation. broad host range plasmids that utilize a unidirectional rolling Cultures were grown in L broth medium with addition of circle replication (rcr) mechanism (13, 14), resembling that of antibiotics according to resistance profiles of strains: ampi- bacteriophage 4X174 (15): the replication protein (Rep) en- cillin (Amp) at 100 pug/ml and spectinomycin (Spc) at 50 coded by the rcr plasmid creates a site-specific nick in the jig/ml. Plasmids were introduced into E. coli strains by the plus strand to initiate replication and attaches to the 5' end of CaC12 transformation method (19). Whole-cell DNA minily- the displaced strand (16). The 3'-OH end produced serves as sates (20) and plasmid DNA (19) were prepared by published a primer for DNA synthesis. Replication intermediates are methods. a-shaped molecules (unpublished data; D. Halpern and L. Oligonucleotides. Three oligonucleotide pairs were synthe- Janniere, personal communication) with less than monomer- sized (by J. Buri, this institute): length tails (15). After one round of replication, Rep nicks a second time at the origin to terminate replication. If termi- x1, S'-AATTCAGCTGCTGGTGGA -3' nation does not occur-due, for example, to a break in the 3'- GTCGACGACCACCTTCGA-S' displaced strand or to a nonattached Rep protein-rcr would x2, 5'-AATTCAGCTGCCACCAGCA -3' continue indefinitely, giving rise to a or molecule with a long 3'- GTCGACGGTGGTCGTTCGA-S' tail. We have identified high molecular weight plasmid spe- Abbreviations: HMW, high molecular weight linear multimer; ss- The publication costs of this article were defrayed in part by page charge DNA, single-stranded DNA; dsDNA, double-stranded DNA; payment. This article must therefore be hereby marked "advertisement" RecBCD Exo, RecBCD exonuclease; rcr, rolling circle replication. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

12073 Downloaded by guest on September 30, 2021 12074 Genetics: Dabert et al. Proc. Nadl. Acad. Sci. USA 89 (1992)

} X X://- Plasmids. Derivatives of the rcr replicon pC194 (21) were used; plasmid pHV950 [4.5 kilobases (kb)] contains the Ampr gene and the rriB site of pBR322 (18), and pHV903 (4.9 kb) MMl\ contains Amp and the pBR322 origin [ref. 14 (described as pHV33APvu)]. These additional functions improve transformation efficiency ofthe pC194 replicon in E. coli (H. te Riele, personal communication). The pBR322 origin present in pHV903 does not affect HMW formation by the pC194 replicon (unpublished data). The X linkers have LfindIII and EcoRI ends and were cloned onto HindIII/EcoRI linearized pHV950 and pHV903 (deleting 25 bp from plasmids). The site .u ofX insertion is 1.5 kb upstream from the plus-strand origins of pHV950 and pHV903. Indcible Replication System. The replication gene of pC194 (rcr rep) was previously isolated and placed under control of the bacteriophage A PL promoter (plasmid pHV980; ref. 22); it was used to conditionally activate the rcr origin in trans (22) (plasmids kindly supplied by M. F. Gros, FIG. 1. (A) Orientation ofV linkers in rcr plasmids. x2 was cloned this institute). To construct target plasmids, aHindIII/EcoRI into all rcr derivatives so that it would be recognized by RecBCD (the fragment containing the 55-bp rcr origin (22) wasjoined to the "packman") entering through the tail of the a-form replication HindIII/EcoRI ends of thermosensitive pSC101 Spcr deriv- intermediate. xl was cloned in the opposite orientation. (B) rcr plasmids containing X produce HMW. Total DNA lysates of strains ative, pGB2ts (23, 24), giving pINDKO (4.1 kb). xl and x2 containing pHV903 derivatives were separated on 0.6% Tris borate/ linkers were cloned into pINDXO =60 bp downstream of the agarose gels and analyzed by Southern hybridization with 32P-labeled rcr origin, resulting in pINDxL and pINDX2, respectively pHV9O3 plasmid DNA used as probe. The X inserts carried by (Figs. 2 and 3). Two helper plasmids derived from plasmid pHV903 are indicated above the lanes (X0, no insertion; X2m, a pSF117 were constructed (Figs. 2 and 3). pSF117 is a pBR322 mutated X2 insertion as described in Materials and Methods). derivative containing the phage A gaim gene under control of Plasmid pHV903 contains rriB, an efficient site for initiation of the A PR promoter and cI857, encoding the A thermosensitive complementary DNA strand synthesis (14); thus, ssDNA was not repressor (25). For one helper 1.8-kb detected on this autoradiograph. HMW comigrates with chromoso- plasmid (Fig. 3), a Sac mal DN4A. At least 10 independent tests were performed, giving I (blunted)/Ssp I fragment of pHV980 containing the PL- comparable results. The DNA forms migrating between supercoiled controlled rcr rep gene (22) was cloned into the unique Sal I (sc) plasmid and HMW (see also Figs. 2, 3, and 5) correspond to (blunted) site of pSF117. For the other (Fig. 2), this same dimeric and relaxed plasmid DNA. 1.8-kb segment containing rcr rep wasjoined to the large Sal I/BamHI fragment ofpSF117; the gam gene is not present in X2 S'-AATTCAGCTGCCACCCGCA -3' this plasmid. Growth conditions in induction experiments are 3'- GTCGACGGTGGGCGTTCGA-S' described in Fig. 2 and 3 legends. Detection of HMW. Whole-cell DNA minilysates were run (X is underlined; the base in boldface type is mutated in the on 0.5% and 0.6% agarose gels prepared in Tris borate/ Xsite). Linker sequences were verified after being cloned into EDTA buffer (19) containing ethidium bromide (0.4 ,ug/ml) the rcr plasmids. x2 and X2m are oriented in all rcr plasmids and electrophoresed at 2.4-2.8 V/cm overnight. Gels were described here for recognition by a RecBCD molecule which treated with 0.1 M HCl and then processed as described for enters through the tail of the a replication intermediate (Fig. Southern hybridization (26). HMW, which comigrates with 1A). xl is in the opposite orientation. chromosomal DNA, was detected with nick-translated or A 1 TARGET: pIND hours 4'OC 34 ( 1 _jII _

HELPER _>rcr

- (Amp rNOP * -)\. [il pBR322 p C1857

FIG. 2. HMW induction. (A) Scheme of target and helper plasmids present in JM101. (B) Cells were grown to midlogarithmic phase in L broth ining Spc (target plasmid resistance) and Amp (helper plasmid resistance) at 280C and shifted to 40"C. Cell samples were removed at the indicated time intervals. Whole-cell lysates, prepared from cultures after adjustment for cell concentrations, were deposited on an agarose gel (DNA of -3 x 107 cells). Hybridization was performed with 32P-labeled pGB2 (target DNA) as probe. Autoradiograph is shown. X inserts carred by the target plasmid are indicated above the lanes, as are the times of induction at 40'C. ssDNA is a product of rcr. sc, Supercoiled. Downloaded by guest on September 30, 2021 Genetics: Dabert et aL Proc. Nadl. Acad. Sci. USA 89 (1992) 12075

A TARGET: pIND HELPER f x/ ) /.! . . i_ S1 f

I gam v ,I,

.0 .t 0

°C WAKUt HMW ACTIVE RecBCD B TEPREPL ICON :x~o x'I X21 28 8 + ***,Q*a _I e*-* * "' 40 rcr * - ** +++

SHIFT 0 + +

FIG. 3. HMW sensitivity to RecBCD enzyme; fate of HMW with and without X after RecBCD is restored. (A) Scheme of target and helper plasmids present in JM101. Cells were grown to midlogarithmic phase in L broth containing Spc and Amp at 280C, shifted to 400C for 70 min (Rep is activated and RecBCD is inactivated, so all rcr plasmids make HMW), and shifted back to 280C (RecBCD regains activity). In these experiments, shiftdown from 40'C to 280C was performed at cell OD600 = 0.8. Cell samples were taken at each stage and treated as described in Fig. 2. (B) Summary of results. $, Measured on target plasmid containing X as indicated; *, rcr is activated by rcr Rep protein provided by helper plasmids; **, RecBCD is inactivated by Gami provided by helper plasmid; + and -, presence and absence of the indicated activity. (C) Gel autoradiograph. X linkers carried by the target plasmid are indicated above the lanes. Samples were taken as follows: lanes 1, at 28"C; lanes 2, after 70 min at 400C; lanes 3-6, after subsequent shiftdown to 280C for 0.5, 1, 2, and 3 hr. respectively. sc, Supercoiled.

random-primed plasmid DNA labeled probes (kits from EMW Formation Depends on rcr and the Orientation -of X. Boehringer Mannheim). To further document that HMW formation depends on X orientation and on rcr, a system was designed in which rcr RESULTS plasmid replication was inducible (Fig. 2A). The target plasmid, pINDxO, consisted of a 0-type thermosensitive vector rcr Plasmids Make HMW in an E. coil recBCD Strain. (pSC101 derivative, pGB2ts) containing the origin of repli- Profiles of plasmids replicating by either a rcr (pHV950 and cation of rcr plasmid pC194. X was inserted into pINDxO in pHV903) or a 0 (pBR322) mechanism were examined in either orientation, giving pINDXl and pINDI2. The helper recombination proficient (AB1157) and isogenic recBCD null plasmid (a pBR322 derivative) encodes the cI857 thermosen- strains (data not shown). Neither 0 nor rcr replicons make sitive repressor and the Rep protein of pC194 (Rep expres- HMW in AB1157, and the 0 replicons make little if any in the sion is under control of the A PL promoter). Cells containing recBCD null strain, as reported (27). However, rcr plasmids a pIND and the helper plasmid were grown at 28TC, and make large amounts of HMW in the recBCD strain. We cultures were shifted to 400(. At 280C, the pINflplasmids are hypothesized that HMW is formed whenever termination maintained by the 0 replicon, and rcr is repressed. At 400C, after a round of rcr plasmid replication is missed but that it the 0 replicon becomes inactive; however, the pC194 rep is degraded by RecBCD Exo in rec+ strains (18). gene present on the helper plasmid is induced and pIND rcr Plasmids with Insertions of X Sequence Produce HMW replicates by rcr, as indicated by ssDNA production (Fig. 2B, in Wild-Type E. coil. Since the X sequence 5'-GCTGGTGG-3' lowest band). No HMW is produced when plasmids replicate is proposed to antagonize DNA degradation by RecBCD (28, by a 0 mechanism. Significant amounts of HMW were 29), we considered that its presence on rcr plasmids would produced by plasmid pINDx2 after 2 hr at 400C, whereas permit HMW formation in wild-type cells. DNA linkers barely detectable amounts were observed for pINJY', and none was produced by pINDx° (Fig. 2B). These experiments containing the X sequence were cloned into rcr plasmid show that rcr is necessary for HMW production and that X pHV903 (referred to here as pHV903xO) such that X was present in either orientation, giving plasmids pHV903Xl and has an orientation-specific effect on the formation of HMW by rcr plasmids. The active X insert is the one recognized by pHV903X2. pHV903x2m contains a mutated X sequence in RecBCD if it entered through the tail of the a replication orientation 2. If RecBCD entered through the tail of the a intermediate (Fig. 1A). It appears that X in the opposite form produced during rcr, it would recognize x2 but not xl orientation is not (or is only weakly) recognized by RecBCD. (Fig. 1A). Large amounts of HMW were present in the strain It is notable that HMW appears only after 2 hr ofinduction, lower amounts were in the strain with containing pHV903x2, whereas rcr is active within 1 hr (as judged by ssDNA pHV903Xl, and none was in those with pHV903X2m or production); these results suggest that HMW initiation may pHV903xO (Fig. 1B). Restriction enzyme and exonuclease be inefficient. analyses indicated that HMW molecules consist of tandem X Blocks RecBCD Degradative Activity on BWMolues head-to-tail linear plasmid repeats (data not shown). We in Vivo. We hypothesized that all rcr plasmids produce HMW conclude that the presence of the X sequence in a rcr plasmid as aberrant replication products that are normally degraded causes the appearance of HMW and that its effect is more in the wild-type host by RecBCD; the X site would block pronounced in pHV9O3x2. HMW degradation by RecBCD. To test this, A Gam was used The HMW produced by pHV903xl might be due in part to to conditionally inactivate RecBCD Exo at 400(. In this low-level recognition by RecBCD of X in the reverse orien- system, the gam gene, controlled by the bacteriophage A PR tation (30) or to nonspecific replication ofpC194 in the wrong promoter (25), is present on the helper plasmid (Fig. 3A), and direction; the small amounts of HMW thus formed could target plasmids are as described above (Figs. 2A and 3A). At accumulate in the culture. 400(, cells containing these plasmids become phenotypically Downloaded by guest on September 30, 2021 12076 Genetics: Dabert et al. Proc. Nat!. Acad. Sci. USA 89 (1992)

RCCBCD Interact with X May Titrate Exo Activity. Pro- * XO tection of %-containing HMW (X-HMW) in the experiment * xI described above could be due to titration ofactive RecBCD by 100 A multiple X sites. This would predict that strains containing 10 X2 would remain O no plasmid X-HMW phenotypically RecBCD Exo- longer than those lacking r-HMW. We used T4 2- phage infection to r. 0.1-I address this question, as cells that express RecBCD degrade T4 2-, while cells devoid of RecBCD allow phage develop- ment (33). Cultures used in the experiment described above 0.1- were also tested for T4 2- phage sensitivity at the time points l used for HMW analysis. A strain containing only the helper 0 30 60 120 180 NO (gam+) plasmid-i.e., not producing HMW-served as con- SHIFT trol, and the recB21C22 mutant was used to enumerate the Time (minutes) after shift-down from 400C to 280C number of phage particles present (Fig. 4). RecBCD Exo is efficiently inactivated by Gam at 40TC as reported (25). After FIG. 4. T4 2- sensitivity of strains as a function of Gami expres- shiftdown, RecBCD is restored at the same rate in strains sion and r-HMW content. Experiment described in Fig. 3 was containing just the helper plasmid or helper plus paiNIY. In performed and 200 ul of cell samples were removed at time points contrast, RecBCD is more slowly restored in cells contaiing indicated in Fig. 3. An additional strain containing only the helper Exo (gam+) plasmid was also processed. Cells were mixed with 2 x 102 pINDx1 or pINDx2. These results indicate that RecBCD to 2 x 104 plaque-forming units (pfu) of T4 2- [pfu determined by activity is diminished through interaction withy. plating on recB21C22 (JC5519) indicator strain]. After a 10-min RecA and x2 Are Required for rcr Paid BMW Accumu- incubation at room temperature, the mixture was plated on soft agar. latlon Only if RecBCD Is Present. 0-type plasmids were Plates were incubated at 280C for all samples except those taken at reported to form HMW in recBCD (or gam+) sbcB strains the end of the Gam induction period at 400C, which were placed at (34). RecA was required in these contexts and was proposed 400C (time 0). pfu for each strain and condition is presented as the to formjoint molecules between a structures (34). We asked percentage of the total pfu determined on the recB21C22 indicator whether rcr plasmid-generated HMW also required RecA. strain (y axis). The time at which samples were removed after temperature shiftdown to 280C is plotted on the x axis. The no shift pHV903,0, pHV903Xl, pHV903X2, pHV903X2m, and sample did not undergo shiftup and remained at 280C. (Inset)% inserts pBR322 were compared for HMW accumulation in AB1157 carried by target plasmids are indicated; no plasmid corresponds to and its recA13, recB21C22, and recAl3 recB21C22 deriva- the strain containing only the helper plasmid. tives (Fig. 5). In AB1157, only X-containing plasmids produce HMW. In the recA strain, none of the plasmids produces RecBCD Exo- (31) (we presume that the relevant activity of HMW. In contrast, all rcr plasmids, even without X, produce Gam is the inactivation of RecBCD rather than the inactiva- HMW in recBCD and in recA recBCD strains (Fig. 5), and tion of SbcC; ref. 32), and all rcr plasmids should produce also in a ArecA strain expressing Gam (data not shown). The HMW, even without aX insert. The fates ofHMW DNA with Oreplicon pBR322 did not produce HMW in any background. then be after RecBCD These results show that RecA is required for HMW accu- and without X could compared activity mulation only in a RecBCD+ context. The nonrequirement of is restored by temperature shiftdown. RecA in recBCD (or gam+) cells in these experiments dis- As expected, all three target plasmids (with and without X) tinguish rcr plasmid- from 6 plasmid-mediated HMW forma- produce HMW when RecBCD inactivation and rcr were tion. induced simultaneously (by Gam and Rep proteins, respectively) for 70 min at 40TC (Fig. 3C, lanes 2). Within 1 hr after shiftdown to 28TC, HMW produced by pINDxO is nearly DISCUSSION totally degraded (XO lanes 3 and 4), reflecting the restoration In1ertio of a X Site into rcr ReulsIn Accumu- of RecBCD activities. In contrast, HMW made by pINDJY lIon ofHMW. We have observed that rcr plasmids produce and pINDx2 remained intact for 3 hr or was degraded ex- HMW in a recBCD mutant strain. A simple model to explain tremely slowly (x1 and x2 lanes 3-6). Our data show that a this observation is that the failure to terminate a round of rcr succession of X sites efficiently prevents RecBCD degrada- generates a a-shaped molecule with a growing tail and that tion of DNA in vivo, surprisingly regardless ofX orientation. RecBCD Exo can degrade the tail. It was proposed that Exo

rccA/.'I A/l' rBU('/(2162_ie Z' X 7- /X2-"pBR X X X C -T /' /B'/ X''1R /1 //, 1p

FIG. 5. RecAandx2arerequiredforrcrplasmid- - HI W HMW formation only ifRecBCD is present. Strains containing the plasmids pHV903X° MO lanes), pHV903X' (xl lanes), pHV903X2 (x2 lanes), pHV903X2m (X2m lanes), or pBR322 (pBR lanes) were grown with selection to midlogarithmic phase, and whole-cell DNA lysates were prepared. DNA _ SPw AP__ samples were separated by agarose gel electropho- a_ resis and analyzed by Southern hybridization with pBR322 DNA used as probe (all plasmids contain segments of pBR322). recB2lC22 strains containing rcr plasmids grow poorly; smearing of the HMW U .i h. profiles may be due to partial cell lysis during growth. sc, Supercoiled. Downloaded by guest on September 30, 2021 Genetics: Dabert et al. Proc. Natl. Acad. Sci. USA 89 (1992) 12077

activity can be blocked by the X sequence (28, 29), and we same conditions as those that rescue A red-gam-. We show that plasmids that carry X form HMW in the presence propose that the presence of a X site protects the oa interme- of RecBCD, whereas those that do not carry X, or that carry diates from RecBCD degradation, thus allowing concatemer an inactive X, do not form HMW. Large amounts of HMW formation in both cases and encapsidation for A. It has long accumulate only if X is oriented for recognition by RecBCD been assumed that A red-gam- phage containing X are entering through the tail ofthe o replication intermediate. We rescued by forming recombinant dimers, which can be pack- suppose that HMW formation by rcr plasmids in recBCD aged (38). Our data imply that, if replication is allowed, mutants, or upon X insertion, occurs by the same mechanism; rescue could also be due to packaging of A concatemers. RecBCD is inactivated by a mutation in the enzyme in one case, and by X interaction (28, 29) in the other. We thank W. Wackernagel for his kind gift of T4 2- phage, and E. Formation of 11MW by aX Insertion Is Inefficient. We have Maguin, M. F. Gros, and R. Devoret for providing strains and noted that inactivation of RecBCD Exo (by Gam) was more plasmids. We are grateful to E. Maguin, P. Duwat, B. Michel, and effective in producing HMW than cloning of x2 on the rcr G. Smith for discussion of this work, and to E. Cassuto, E. Maguin, plasmid (compare results described in Figs. 2 and 3). This D. Petranovic, F. Stahl, A. Kuzminov, and the unknown reviewers observation adds to previous in vivo evidence (35) that for very thoughtful criticisms of this manuscript. RecBCD may not always recognize a properly oriented X; that the RecBCD-x encounter is inefficient. Recent in 1. Buttin, G. & Wright, M. (1968) Cold Spring Harbor Symp. Quant. i.e., Biol. 33, 53-66. vitro experiments (36) show that X cutting is -40%o efficient, 2. Clark, A. J. (1973) Annu. Rev. Genet. 7, 67-86. consistent with the interpretation of in vivo results. 3. Telander-Muskavitch, K. M. & Linn, S. (1981) in The Enzymes, ed. A Succession of X Sites on BMW in Either Orientation Boyer, P. C. (Academic, New York), Vol. 14, pp. 233-250. Protects Molecules from Degradation by RecBCD. Once 4. Taylor, A. F. (1988) in Genetic Recombination, eds. Kucherlapati, HMW is formed by conditional inactivation of RecBCD, the R. & Smith, G. R. (Am. Soc. Microbiol., Washington), pp. 231-263. presence of a succession of X sites in either orientation 5. Ponticelli, A. S., Schultz, D. W., Taylor, A. F. & Smith, G. R. protects it from RecBCD degradation (Fig. 3 B and C). In (1985) Cell 41, 145-151. 6. Taylor, A. F., Schultz, D. W., Ponticelli, A. S. & Smith, G. R. contrast, HMW DNA devoid of X sites is rapidly degraded. (1985) Cell 41, 153-163. These results show that X effectively protects DNA from 7. Roman, L. J., Dixon, D. A. & Kowalczykowski, S. C. (1991) Proc. degradation. Natl. Acad. Sci. USA 88, 3367-3371. To explain why HMW is protected by X regardless of 8. Dixon, D. A. & Kowalczykowski, S. C. (1991) Cell 66, 361-371. orientation, we must assume that the long-tailed or forms 9. McMilin, K. D., Stahl, M. M. & Stahl, F. W. (1974) Genetics 77, often break and that resulting linear fragments are susceptible 409-423. to RecBCD nuclease from both ends. In a pro- 10. Lam, S. T., Stahl, M. M., McMilin, K. D. & Stahl, F. W. (1974) previously Genetics 77, 425-433. posed model, RecBCD loses Exo activity upon encounter 11. Stahl, F. W., Crasemann, J. M. & Stahl, M. M. (1975) J. Mol. Biol. with X (28, 29). If RecBCD Exo activity is diminished after X 94, 203-212. encounter, then multiple X sites present on HMW could 12. Stahl, F. W. & Stahl, M. M. (1977) Genetics 86, 715-725. titrate the enzyme present in low amounts in cells (10-15 13. te Riele, H., Michel, B. & Ehrlich, S. D. (1986) Proc. Natl. Acad. molecules per cell; ref. 4). Our results support this interpre- Sci. USA 83, 2541-2545. tation; after inactivation by Gam, RecBCD Exo activity is 14. te Riele, H., Michel, B. & Ehrlich, S. D. (1986)EMBOJ. 5, 631-637. restored more slowly in the presence of X-containing HMW 15. Gros, M.-F., te Riele, H. & Ehrlich, S. D. (1987) EMBO J. 6, than in the 3863-3869. presence ofy0-HMW (Fig. 4). Recent in vitro data 16. Koepsel, R. R., Murray, R. W., Rosenblum, W. D. & Khan, S. A. showing a reduction in all RecBCD activities after X cutting (1985) Proc. Natl. Acad. Sci. USA 82, 6845-6849. (36) may also be interpreted to agree with this model. 17. Gruss, A. & Ehrlich, S. D. (1988) J. Bacteriol. 170, 1183-1190. Does RecA Help Protect the Ends of HMW from Degrada- 18. Dabert, P., Ehrlich, S. D. & Gruss, A. (1992) J. Bacteriol. 174, tion? In the presence of RecBCD, RecA is required for a rcr 173-178. to HMW. ifRecBCD 19. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular plasmid containingV produce However, Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold is absent, RecA is not required, and any rcr plasmid produces Spring Harbor, NY). HMW, suggesting that RecA is not needed to initiate HMW 20. Projan, S., Carlton, S. & Novick, R. P. (1983) Plasmid 9, 182-190. formation. Rather, RecA may help protect HMW from Exo 21. Horinouchi, S. & Weisblum, B. (1982) J. Bacteriol. 150, 815-825. degradation. Even if a first RecBCD Exo is inactivated by 22. Gros, M.-F., te Riele, H. & Ehrlich, S. D. (1989) EMBO J. 8, encountering a X site on HMW, a second RecBCD molecule 2711-2716. load on the end after X To prevent 23. Clerget, M. (1991) New Biol. 3, 780-788. might generated cutting. 24. Churchward, G., Belin, D. & Nagamine, Y. (1984) Gene 31, reloading, RecA could bind ssDNA frayed ends produced by 165-171. RecBCD unwinding past the X site; in vitro, RecA binding to 25. Friedman, S. A. & Hays, J. B. (1986) Gene 43, 255-263. ssDNA ends of a dsDNA molecule prevented degradation by 26. Southern, E. (1975) J. Mol. Biol. 98, 503-517. RecBCD (37). Since RecA also promotes homologous pair- 27. Niki, H., Ogura, T. & Hiraga, S. (1990) Mol. Gen. Genet. 224, 1-9. ing, it could protect HMW ends from RecBCD reloading by 28. Thaler, D., Sampson, E., Siddiqui, I., Rosenberg, S., Thomason, a a L., Stahl, F. & Stahl, M. (1989) Genome 31, 53-67. mediating synapse with second DNA molecule (8). The 5- 29. Rosenberg, S. & Hastings, P. (1991) Biochimie 73, 385-397. to 10-fold stimulation of recombination by X (9-12, 38) 30. Stahl, F. W., Kobayashi, I. & Stahl, M. M. (1983) in Mechanisms suggests that this step occurs at some frequency. In our of DNA Replication and Recombination, ed. Cozzarelli, N. R. system, we propose that recombination may be a conse- (Liss, New York), pp. 773-783. quence rather than a cause of HMW production. To test this, 31. Murphy, K. (1991) J. Bacteriol. 173, 5808-5821. we are HMW formation in strains defective at 32. Kulkarni, S. K. & Stahl, F. W. (1989) Genetics 123, 249-253. examining 33. Oliver, D. & Goldberg, E. (1977) J. Mol. Biol. 116, 877-881. different steps of recombination. 34. Cohen, A. & Clark, A. J. (1986) J. Bacteriol. 167, 327-335. Parallels Between rcr Plasmid I1MW Formation and A 35. Stahl, F. W., Thomasson, L. C., Siddiqi, I. & Stahl, M. M. (1990) red-gam- Concatemerization. Late replication of A is similar Genetics 126, 519-533. to replication of rcr plasmids in that both involve a interme- 36. Taylor, A. F. & Smith, G. R. (1992) Proc. Natl. Acad. Sci. USA 89, diates. In the case of A red-gam- (XO), these intermediates 5226-5230. are presumably degraded by RecBCD and phage produce tiny 37. Williams, J. G. K., Shibata, T. & Radding, C. M. (1981) J. Biol. Chem. 256, 7573-7582. plaques (see ref. 38 for review). The A red-gam- can be 38. Smith, G. R. (1983) Lambda II, eds. Hendrix, R. W., Roberts, rescued if it is propagated in recBCD or recA recBCD strains J. W., Stahl, F. W. & Weisberg, R. A. (Cold Spring Harbor Lab., or ifit contains aXsite. rcr plasmids produce HMW under the Cold Spring Harbor, NY), pp. 175-209. Downloaded by guest on September 30, 2021