Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6445-6449, October 1985 Biochemistry

Cleavage of cruciform DNA structures by an activity from (DNA-protein interactions/ sequences/Holliday junctions/mechlorethamine) STEPHEN C. WEST AND ANN KORNER Departments of Molecular Biophysics and Biochemistry and of Therapeutic Radiology, Yale University, P.O. Box 6666, New Haven, CT 06511 Communicated by I. Robert Lehman, June 3, 1985

ABSTRACT Protein extracts from Saccharomyces cerevi- e siae have been fractionated to reveal a nuclease activity that cleaves cruciform structures in DNA. Negatively supercoiled that contain inverted repeats that are extruded into cruciform structures have been used as DNA substrates. The sites of cleavage of pColIR215 DNA are located within the extruded cruciform stems and are symmetrically opposed to each other across the cruciform junction. Neither relaxed duplex DNA nor single-stranded DNA serve as substrates. The native molecular weight of the activity was estimated to be =200,000 by gel filtration.

Recent studies of plasmids that contain palindromes have (a) cut at (b) cut at shown that inverted repeat sequences are extruded in vitro to e-e a-c or b-d form cruciform structures and, thus, reduce the free energy of negative supercoiling (1-3). Two classes of enzymes have been shown to cleave plasmids that contain cruciform struc- tures. The first includes a variety of single-strand nucleases, such as S1 nuclease, that nonspecifically cleave the single- stranded of the FIG. 1. Two alternative mechanisms for cleavage of cruciform hairpin loops cruciform (see Fig. 1, path a) DNA. Path a shows nicking by a single-strand nuclease (e.g., S1 (4-6). The enzymes of the second class, which includes T4 nuclease) at each hairpin loop to form linear duplex DNA. Path b endonuclease VII (7) and T7 endonuclease I (8, 9), show a shows that cleavage close to the base of the cruciform junction by a more specific interaction and cleave diagonally across the resolving enzyme (e.g., T4 endonuclease VII) produces a linear cruciform junction (see Fig. 1, path b) (2, 10, 11). duplex with hairpin termini and single-strand nicks. In this diagram, The local DNA structure around a cruciform junction is the size of the cruciform is exaggerated and negative supercoiling is indistinguishable from that of a (2, 10), a omitted. central intermediate in the process of (12-15). Therefore, plasmids that contain inverted repeat 3H]thymidine or 32Pi and prepared as described (17). All DNA sequences may be useful as probes to assay cell extracts for concentrations are expressed in moles of residues. enzymes that specifically recognize and cleave Holliday Enzymes, Materials, and Buffers. Purified T4 endonuclease junctions. VII (200 units/,l) was generously provided by B. Kemper. In this paper, we describe studies with a partially purified Mechlorethamine, phenylmethylsulfonyl fluoride, and fraction from Saccharomyces cerevisiae that cleaves phosphocellulose were obtained from Sigma, DEAE-Bio-Gel cruciform junctions in vitro. The activity cannot be detected A and Bio-Gel A-0.5m were from Bio-Rad, Zymolyase-20T in crude extracts and only becomes apparent during protein was from Miles, and single-stranded DNA (ss DNA)-agarose fractionation. The sites of cleavage of DNA are was from Bethesda Research Laboratories. Buffer R was 20 shown to be located within inverted repeat sequences and mM TrisHCl, pH 7.5/1 mM EDTA/0.5 mM dithiothrei- occur at regions that are symmetrically opposed across the tol/10% glycerol/0.1 mM phenylmethylsulfonyl fluoride. cruciformjunction. As yet, we have been unable to purify the Buffer P was the same except Tris buffer was replaced with activity to homogeneity. However, since the activity appears 20 mM sodium phosphate (pH 6.8). to provide the first example ofa eukaryotic nuclease that acts Purification of Cruciform Cleavage Activity. Four liters of on cruciform junctions in duplex DNA, we feel that its S235/SR154-6C diploid cells were grown in YPD (yeast demonstration is of sufficient interest to warrant this prelim- extract/peptone/dextrose) broth with aeration to 1 x 107 cells inary report. per ml, mechlorethamine was added at 20 ,g/ml, and growth was continued for 3 hr. The cells were harvested by centrifu- MATERIALS AND METHODS gation, washed and resuspended in 100 ml of 50 mM Tris HCl, Strains, Plasmids, and DNA. Yeast strains S235 (a his3-11, pH 7.5/10o sucrose/1 mM EDTA, and stored at -80°C. When -15 leu2-3, -112 cani) and SR154-6C (a ade2-1 trpl-J) were required, cells were thawed, and all subsequent procedures provided by S. Roeder. The plasmids pColIR215 (5) and pUC7 were performed at 0°C. For lysis, we added 6.2 ml of5 M NaCl, (16) were grown in HB101 recA-. Covalently 10 ml ofZymolase at 20 mg/ml, and 100 ,ul of mercaptoethanol closed plasmid DNA was uniformly labeled with [methyl- and incubated at 0°C. After 30 min, 120 1,u of 0.1 M phenylmethylsulfonyl fluoride and 1.2 ml of 10% Brij 58 were The publication costs of this article were defrayed in part by page charge added, and incubation was continued for 20 min. Lysis was payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: ss DNA, single-stranded DNA; bp, base pair(s). 6445 Downloaded by guest on September 26, 2021 6446 Biochemistry: West and Komer Proc. Natl. Acad. Sci. USA 82 (1985) monitored by light microscopy. Cell debris was removed by moved the negative supercoiling required for cruciform centrifugation at 35,000 rpm in a Beckman 42.1 rotor. extrusion. Third, proteolytic activities affected protein sta- DNA was removed from the clear supernatant (fraction I) by bility. However, we did observe an activity specific for passage through a 30-ml DEAE-Bio-Gel A column equilibrated cruciform DNA after fractionation on a DNA-agarose col- with buffer R containing 0.3 M NaCl. The flow-through was umn. This activity was further purified by using phosphocel- precipitated with 0.5 g ofammonium sulfate per ml and dialyzed lulose and DEAE-Bio-Gel to produce fraction IV as de- against buffer P to produce fraction II. Fraction II was loaded scribed. Fraction IV did not contain any exonuclease activity onto a 50-ml ss DNA-agarose column equilibrated with buffer as determined by using 5'- or 3'-end-labeled linear duplex P and was eluted with a 0-0.25 M NaCl gradient in buffer P. DNA. For convenience, the endonuclease activity in fraction Fractions were immediately assayed for the cleavage of IV will be referred to as X nuclease. pColIR215 DNA. The activity specific for cruciform DNA In an identical preparation from which mechlorethamine usually was eluted between 0 and 50mM NaCl and was dialyzed treatment of the cells was omitted, the cruciform-specific against buffer R containing 50 mM NaCl. Fraction III (50 ml) activity was detected at a lower level. Therefore, all subse- was then passed through a 5-ml column of phosphocellulose quent preparations were made from treated cultures. directly onto a 2-ml DEAE-Bio-Gel A column. The activity was Approximately 25% of supercoiled pColIR215 was con- eluted from the DEAE column with a0.05-0.4 M NaCl gradient verted to a linear form by incubation with fraction IV (Fig. 2, and active fractions, which eluted at about 0.25 M NaCl, were lane b). When relaxed by topoisomerase treatment, to gen- pooled and stored in small aliquots at -800C (fraction IV). The erate topoisomers oflow linking number that are incapable of protein concentration of fraction IV was too low to measure. cruciform extrusion, pColIR215 failed to be a substrate for Preparations have shown no loss of activity over a period of 1 the enzyme (Fig. 2, lane d). X nuclease did not degrade year when stored at -800C. 4X174 ss DNA (Fig. 2, lane f) or cut linear duplex DNA of Reactions and Assays. Cleavage reactions (30 Al) generally pColIR215 (not shown), as analyzed by gel electrophoresis. contained 28 ,uM plasmid DNA in 50mM Tris-HCl, pH 8.5/10 Cruciform Structures Are the Targets for Cleavage. The mM MgCl2/i mM dithiothreitol/100 gg of bovine serum approximate site of cleavage of supercoiled pColIR215 DNA albumin per ml/0.5 pk1 of fraction IV or 100 units of T4 endo was determined by treating 32P-labeled plasmid with fraction VII. Reactions were incubated at 370C for 60 or 90 min. They IV, followed by restriction analysis using single-site en- were stopped, and the products were deproteinized by the zymes. In each case, two major fragments were produced, addition of EDTA and NaDodSO4 to 50 mM and 1%, indicating that X nuclease cleaved each plasmid molecule respectively. In experiments with pUC7, it was necessary to once (Fig. 3, lanes c-e). When we treated pColIR215 with T4 preincubate the plasmid at 60'C for 2 hr in 0.2 M NaCl to endonuclease VII and the same restriction enzymes, frag- allow extrusion of the large cruciform (11). pColIR215 plas- ments of similar length were produced (Fig. 3, lanes g-i). mid DNA did not require pretreatment. Since T4 endonuclease VII cleaves pColIR215 at the site of The assay for single-strand nuclease activity measures the the major inverted repeat (10, 19), these results indicate that reduction in the number of infective centers obtained in X nuclease also cleaves at, or close to, this sequence. In transfection experiments after treatment of 4X174 ss DNA addition to the major fragments, we also observed the with various dilutions of enzyme. 4X174 ss DNA (28 ILM) formation of pairs of minor fragments, which result from was incubated with fraction IV or S1 nuclease for 60 min at cleavage by either X nuclease or T4 endonuclease VII at the 370C in the standard reaction buffer or 50 mM sodium minor inverted repeats of pColIR215, which are the natural acetate/10 mM zinc sulfate/150 mM sodium chloride for S1 of pBR322 (10, 18). nuclease. The reaction was stopped by the addition ofexcess palindromes EDTA, and proteins were inactivated by heating at 759C for Other plasmids that contain cruciforms were also substrates 10 min. Then 10-,/1 samples were added to 200 41. of for the nuclease activity in fraction IV. For example, pUC7 CaCl2-treated E. coli cells and incubated at 0C for 30 min, contains a perfect 48-bp cloned palindrome (16). When treated followed by a heat pulse of 3 min at 420C. The mixture then with fraction IV, we observed cleavage that was located by was returned to ice, diluted with Luria broth, and plated restriction mapping to this site (data not shown). Similar results immediately to determine the number of infected cells. were observed by using pBR322 DNA, which contains three The buffer system for neutral agarose gels was 40 mM palindromes 23 to 26 bp long (10) (data not shown). Althoughthe Tris-HCl, pH 7.8/5 mM sodium acetate/1 mM EDTA and for inverted repeats in pColIR215, pUC7, and pBR322 are unre- polyacrylamide gels was 89 mM Tris base/89 mM boric lated in sequence, they were cleaved with equal efficiency by acid/2 mM EDTA. In addition, the polyacrylamide sequenc- ing gels contained 7M urea, and the DNA samples were Fraction IV denatured by heating at 950C for 2 min in 95% formamide/20 mM EDTA. RESULTS Preparation of Cruciform-Specific Fraction. Protein ex- tracts were prepared from exponentially growing diploid Open circles- cerevisiae that had been treated with Linears- cultures of S. -ss DNA mechlorethamine (nitrogen mustard), a DNA-damaging agent that is known to increase the frequency of mitotic Supercoiled - recombination (18). The DNA substrate in the cleavage assay circles was pColIR215 (5, 10), a recombinant plasmid that contains a 31-base-pair (bp) palindrome from ColEl with 5 bp of nonrepeated sequence at its center. When negatively super- a b c d e f inverted to form a coiled, the plasmid extrudes this repeat FIG. 2. Cleavage of supercoiled pColIR215 DNA by fraction IV. cruciform structure. DNA was incubated with or without fraction IV as described, and the Preliminary experiments to search for a cruciform-specific products were visualized by ethidium bromide staining of a 1% activity revealed three problems. First, the extracts con- agarose gel. Lanes: a and b, supercoiled plasmid DNA; c and d, tained high levels of single-strand endonuclease activity that plasmid DNA relaxed by topoisomerase I; e and f, 4X174 ss DNA. cleaved the substrate DNA. Second, a topoisomerase re- Fraction IV was present in the reactions of lanes b, d, and f. Downloaded by guest on September 26, 2021 Biochemistry: West and K6mer Proc. Natl. Acad. Sci. USA 82 (1985) 6447 major I.R. turation, we tested for cutting of relaxed plasmids that EcoR /,TI contained thymine dimers, psoralen adducts, or mismatched BamHI bases. However, we were unable to demonstrate cleavage of PstI SilI these substrates, which contain unpaired regions in duplex DNA (data not shown). pColIR215 Requirements for Cleavage. Cleavage ofcruciform DNA by fraction IV required the presence of magnesium as the minor I.R. divalent cation (data not shown). Concentrations between 5 Av I and 10 mM MgCl2 were optimal. Neither Ca2+, Co2+, nor Mn2+ could substitute for Mg2+. We have not observed a requirement for any additional cofactors. Fraction IV was Fraction =2 T4 endo= active over a broad range ofpH, with pH 8.5 as optimum. The Ava I + +- activity was stable during incubation in 50 mM Tris HCl (pH PstI iI + SalT + 7.5) at 0C or 370C for 1 hr. However, activity was lost after heating at 650C for 10 min or by treatment with 1 mg of a b cd e f g h i proteinase K per ml and 1% NaDodSO4. The activity was Origin- insensitive to RNAse treatment. The protein concentration of fraction IV was too low to allow a determination of purity by NaDodSO4 gel electro- phoresis. When analyzed by gel filtration through a Bio-Gel Nicked circle- A-0.5m sizing column in R buffer supplemented with 100 mM NaCl, active fractions were eluted with a molecular weight of Linear - -200,000 compared with standard markers (thyroglobulin, Superhelical - 670,000; gamma globulin, 158,000; ovalbumin, 44,000; circle myoglobin, 17,000; cyanocobalamin, 1,350). Fraction IV Introduces Staggered Cuts in the Inverted Repeat Sequence. The results are consistent with the nuclease activity in fraction IV cutting cruciform DNA not only at positions a and c or b and d (Fig. 1), but also at positions a and d or b and c. To determine the relative frequency of cutting at these positions, pColIR215 was incubated with fraction IV and subsequently digested with Taq I, which cuts 0 90 bp from the center of the inverted repeat. The small DNA fragments produced in the reaction were analyzed by poly- FIG. 3. Specific cleavage of the pColIR215 cruciform by fraction acrylamide gel electrophoresis. Sequence data (10) indicate IV or T4 endonuclease VII. Uniformly 32P-labeled plasmid DNA was that cleavage at positions a and d or b and c should, together treated with either fraction IV (lanes b-e) or endonuclease VII (lanes with Taq I digestion, produce two new fragments of lengths f-i) for 60 min. Aliquots were withdrawn and digested with Ava I, Pst 74 and 105 bp (i.e., differing in size by the length of the I, or Sal I as indicated and were analyzed by electrophoresis through inverted repeat). In contrast, cleavage at a and c or b and d 1.2% agarose followed by autoradiography. The restriction map should produce only one new restriction fragment. Fig. 4, shows the position of the major inverted repeat between EcoRI and lane b, shows one new band, which migrated close to an 82-bp the indicated Taq I site. Other Taq I sites present in the plasmid DNA marker restriction fragment. This fragment was absent in are not indicated on this diagram. The smaller boxes, designated "minor I.R.," represent the natural inverted repeats ofpBR322 (10). control reactions from which fraction IV was omitted (data not shown). The other band visible in lane b ofFig. 4 was seen fraction IV. This result indicates that the nuclease is specific for a b structure rather than for sequence. Moreover, since negative k ' supercoiling is required for cruciform extrusion and forcleavage 311- of these by fraction IV, the structure recognized by the nuclease activity is likely to be some feature of the cruciform. 2499- Since inverted repeat sequences in supercoiled plasmids are strong sites for attack by single-strand nucleases (5), we 200- _*Um compared the single-strand nuclease activities of fraction IV 151 - obw".1"dw and S1 nucfease. As an assay, we used the loss of infectivity 140- "Wowmw of 4X174 ss DNA after the introduction of one nick. This extremely sensitive test showed that the amount of fraction 1 1 8- IV (0.5 ,l) that was sufficient to promote the maximal amount 100- of cleavage of plasmid DNA that contained a cruciform, 82- cleaved only 12% ofphage ss DNA in 1 hr at 37°C. In a control experiment, we determined the amount of S1 nuclease 66 - wm required to linearize :30o of supercoiled pColIR215. This concentration of S1 nuclease (0.1 unit) totally degraded the 4X174 ss DNA, and no infective centers were observed. These data show that fraction IV is substantially free of single-strand nuclease activity. Therefore, it is unlikely that FIG. 4. Fine mapping of the cleavage site in pColIR215. Lanes: the specific cleavage ofcruciform DNA by fraction IV results a, size markers in bp (Hinfl digest of#X174 duplex DNA); b, plasmid from cutting of the single-stranded hairpin loops of the DNA treated with fraction IV followed by Taq I digestion. All DNA cruciform structure (position e-e in Fig. 1, path a). was 3'-end-labeled with 32P and analyzed by electrophoresis through Since it is possible that fraction IV might be specific for an 8% polyacrylamide gel (nondenaturing) followed-by autoradi- duplex DNA substrates that contain regions of local dena- ography. Downloaded by guest on September 26, 2021 6448 Biochemistry: West and Korner Proc. Natl. Acad. Sci. USA 82 (1985) in control reactions and corresponds to an internal 141-bp between the C and T residues, and the second major site of Taq I fragment of pColIR215 (positions 1126-1267 of the cleavage occurred between T and A to produce a fragment pBR322 sequence; ref. 20). These data indicate that cleavages one nucleotide shorter. Other, more minor, sites of cleavage at a and d or b and c occur rarely under our conditions. were observed close to the center of the inverted repeat. No To determine the precise sites of cleavage relative to the cleavage of significance was introduced on the 5' side of the cruciform structure, we treated pColIR215 DNA with frac- inverted repeat, which would have produced fragments -20 tion IV followed by EcoRI digestion. The FcoRI termini were longer. 3'-end-labeled with 32p, and the DNA was denatured and run We also determined the site ofcleavage in the other strand. on a sequencing gel. To provide sequence markers, the pColIR215 that had been treated with fraction IV was cut 190-bp EcoRI-Taq I fragment of pColIR215 was purified, with Taq I, and the Taq I terminus was 3'-end-labeled. As 3'-labeled at the EcoRI terminus, and subjected to markers, we produced a Maxam-Gilbert sequencing ladder Maxam-Gilbert chemical degradation (21). By taking into from a 190-bp EcoRI-Taq I fragment of pColIR215 that was account that one nucleoside was chemically removed by the 3'-labeled at the Taq I terminus. The sequencing gel of Fig. Maxam-.Gilbert method, the results in Fig. 5 indicate that 6 shows that the sites of cleavage in this strand are similar to cutting took place within the C-T-A-G sequence on the 3' side those observed with the first strand. Again, cleavage oc- of the inverted repeat. The major site of cleavage occurred curred predominantly in the C-T-A-G sequence, and the major site of cleavage was between the C and T residues. G G/A T/ C C These results show that the major sites of cleavage by C fraction IV occur in symmetrically related positions about the T inverted repeat sequence. *4.z4A A T G G/A T/C C .,G TT I ~G IT .z

A G T _.I G. T .s A C ii A c T T A C G L9J C C A A A -. T T c .

T A A T A T C T A A A A

T

FIG. 5. Sites of cleavage of pColIR215 by fraction IV. Plasmid DNA, treated with fraction IV and digested with EcoRI, was 3'-labeled at the EcoRI termini. Fragments were run on an 8% sequencing gel flanked by chemical degradation ladders obtained FIG. 6. Sites of cleavage by fraction IV. Plasmid DNA treated from an EcoRI-Taq I pColIR215 fragment 3'-labeled at the EcoRI with fraction IV were digested with Taq I and 3'-labeled at the Taq terminus. The sequences shown on the right were taken from ref. 10 I termini. The fragments obtained were run on a sequencing gel and were in close agreement with those obtained here except for the flanked by chemical degradation ladders obtained from an region on the 5' side (upper) of the inverted repeat, where sequence EcoRI-Taq I pColIR215 fragment 3'-labeled at the Taq I terminus. reading is made difficult because the palindrome alters fragment The sequences shown on the right were taken from ref. 10 and were mobility. as described in the legend to Fig. 5. Downloaded by guest on September 26, 2021 Biochemistry: West and K6rner Proc. Natl. Acad. Sci. USA 82 (1985) 6449

pColIR21 5 a number ofreasons. (i) The DNA connections at thejunction of a cruciform structure are geometically equivalent to those A A T of a Holliday structure (2, 10, 22). (it) Phage enzymes that A G cleave cruciform structures also cleave Holliday structures in C=G C=G vitro (2, 10, 11) and are thought to be involved in the T=A resolution of branched structures in vivo (2, 7, 23). (iii) The A=T A=T instability of plasmids that contain long inverted repeats, at C=G least in E. coli, is suggestive of destructive processing by G=C A=T Holliday-resolving enzymes (5, 24, 25). T=A While fraction IV has been shown to cleave cruciform C= structures, the precise nature of the DNA structure recog- EcoRI C=G TaqI T=A nized by the activity has not been determined. Indeed, G=C although cleavage occurs within stem sequences, it is not at AAA C :AA-----* *-----TTTr G,TT------present clear whether a hairpin loop or the base of the C=G cruciform is the structural feature recognized by the activity. A=T G=C While it may be tempting to speculate that the activity might G=C be capable of resolving Holliday structures, and none of our =T 4=A present data rules out this possibility, caution must be =G exercised until the activity has been purified extensively and G=C T=A tested on other DNA substrates such as figure-8 DNA T=A molecules made by RecA protein (17) and X-forms prepared A=T G=C by hybridizing specific DNA fragments (2). G=C T C I would like to thank Prof. Paul Howard-Flanders for his interest T A T and helpful criticisms. Dr. Borries Kemper generously provided the T4 endonuclease VII, and Drs. David Lilley and Bernard deMassy provided the plasmids used in this study. The work was supported by FIG. 7. The major cruciform structure of pColIR215 showing the U.S. Public Health Service Grants GM11014, CA26763, and two symmetrically related regions of cleavage by fraction IV (ar- AM09397 to Prof. Howard-Flanders. rows). The major sites of cleavage between C and T residues are indicated with a solid arrowhead; 3' termini are indicated with an 1. Hsieh, T. & Wang, J. C. (1975) Biochemistry 14, 527-535. asterisk. 2. Mizuuchi, K., Kemper, B., Hays, J. & Weisberg, R. A. (1982) Cell 29, 357-365. DISCUSSION 3. Gellert, M., Mizuuchi, K., O'Dea, M. H., Ohmori, H. & In this paper we have demonstrated the presence of a novel Tomizawa, J. (1978) Cold Spring Harbor Symp. Quant. Biol. 43, 35-40. endonuclease in a partially purified fraction from S. cerevi- 4. Lilley, D. M. J. (1980) Proc. Natl. Acad. Sci. USA 77, siae. Using plasmid substrates that contain inverted repeats 6468-6472. that are extruded into cruciform structures, we have shown 5. Lilley, D. M. J. (1981) Nucleic Acids Res. 9, 1271-1289. that the nuclease activity catalyzes cleavage close to the 6. Panayotatos, N. & Wells, R. D. (1981) Nature (London) 289, cruciform junction. Studies of the cleavage sites of 466-470. pColIR215 indicate that nicks are introduced into the two 7. Kemper, B. & Garabett, M. (1981) Eur. J. Biochem. 115, extruded cruciform arms at the positions indicated in Fig. 7. 123-131. The sites ofcleavage ofpColIR215 by T4 endonuclease VII 8. Center, M. & Richardson, (1970) J. Biol. Chem. 245, have been characterized in detail (10). Lilley and Kemper 6285-6291. 9. Sadowski, P. (1971) J. Biol. Chem. 246, 209-216. showed that endonuclease VII introduced cleavages into the 10. Lilley, D. M. J. & Kemper, B. (1984) Cell 36, 413-422. cruciform arms at positions two to three nucleotides from the 11. deMassy, B., Studier, F. W., Dorgai, L., Appelbaum, E. & base of thejunction. Cleavages occurred in each strand at the Weisberg, R. A. (1984) Cold Spring Harbor Symp. Quant. 5' end of the inverted repeat. Using the same plasmid, we Biol. 49, 715-726. have shown that the cuts introduced by fraction IV also occur 12. Holliday, R. (1964) Genet. Res. 5, 282-304. in the cruciform arms. However, in this case, cuts occur at 13. Meselson, M. M. & Radding, C. M. (1975) Proc. NatI. Acad. positions four to six bases from the base ofthejunction, at the Sci. USA 72, 358-361. 3' side of the inverted repeat (Fig. 7). The major sites of 14. Radding, C. M. (1982) Annu. Rev. Genet. 16, 405-437. cleavage within these regions occur between C and T resi- 15. Howard-Flanders, P., West, S. C. & Stasiak, A. (1984) Nature (London) 309, 215-220. dues (Figs. 5 and 6). The variability observed at the site of 16. Vieira, J. & Messing, J. (1982) Gene 19, 259-268. cleavage may be inherent to the nuclease activity or may be 17. West, S. C., Countryman, J. K. & Howard-Flanders, P. (1983) a consequence of incomplete extrusion of the cruciform. At Cell 32, 817-829. the present time, we cannot distinguish between these or 18. Zimmermann, F. K. (1971) Mutat. Res. 11, 327-337. other possibilities. 19. Kemper, B., Jensch, F., Depka-Prondzynski, M., Fritz, H., The two regions ofcleavage ofpColIR215 DNA by fraction Borgmeyer, U. & Mizuuchi, K. (1984) Cold Spring Harbor IV are symmetrically opposed across the cruciform junction Symp. Quant. Biol. 49, 815-825. (Fig. 7). In this respect, the activity appears similar to T4 20. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 77-90. endonuclease VII and T7 endonuclease I (2, 10, 11). By 21. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, contrast, other enzymes, such as S1 nuclease, that have been 499-560. used in vitro as probes for cruciform structures cleave in the 22. Mizuuchi, K., Mizuuchi, M. & Gellert, M. (1982) J. Mol. Biol. single-stranded regions of the hairpin loops (4, 6). 156, 229-243. When this study was undertaken, our objective was to 23. Kemper, B. & Brown, D. T. (1976) J. Virol. 18, 1000-1015. 24. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., search for an enzyme capable of resolution of Holliday Heyneker, H. L., & Boyer, H. W. (1977) Gene 2, 95-113. structures, a central intermediate in genetic recombination. 25. Sadler, J. R., Tecklenberg, M. & Betz, J. L. (1980) Gene 8, We chose to use cruciform structures as DNA substrates for 279-300. Downloaded by guest on September 26, 2021