Action of RecBCD enzyme on cruciform DNA

J. Mol. Biol. (1990) 211, 117-134

Action of RecBCD Enzyme on Cruciform Andrew

DNA

F. Taylor and Gerald R. Smith

Fred Hutchinson Cancer Research Center 1124 Columbia Street Seattle, WA 98104, U.S.A. (Received 18 October 1988, and in revised form 18 August 1989) W’e tested the hypothesis that RecBCD enzyme of Escherichia coli resolves pre-existing Holliday recombination intermediates by examining the action of the purified enzyme on an open-ended DNA cruciform with limited ability to branch migrate. The enzyme cleaved two strands of the cruciform near its base to produce “recombinant” products, with a marked bias in the direction of cleavage. The two nicks necessary to cleave the cruciform were made separately. Cruciforms whose four termini were blocked by synthetic hairpin-shaped oligonucleotides were not detectably nicked by the enzyme. With one terminus open the enzyme made a nick at the base of the cruciform but not a double-strand cut. With two or more termini open the enzyme made double-strand cuts. We infer that RecBCD enzyme molecules must enter the termini of duplex DNA and approach the cruciform from more than one direction in order to cleave it into recombinant products. Previous results on RecBCD-mediated recombination between phage A and Ldv imply that intracellular RecBCD enzyme can approach pre-existing Holliday junctions from only one direction. We infer that intracellular RecBCD enzyme cannot cleave pre-existing Holliday junctions int’o recombinants and suggest that the enzyme may cleave Holliday junctions in whose formation it participates.

1. Introduction The principal route for the homologous recombination of DNA entering Escherichia coli is the RecBCD pathway, named after the only known enzyme specific to the pathway. In this paper we examine the action of purified RecBCD enzyme on DNA substrates that resemble the Holliday junction?, a postulated intermediate in many models of genetic recombination (Holliday, 1964). We test the hypothesis that one of the activities of RecBCD enzyme in recombination is to cleave such recombination intermediates into separate recombinant DNA molecules. The Holliday junction (Fig. l(a)) is a fourstranded DNA structure in which two parental DNA substrates are joined at a region of homology by the swapping of single strands. The structure was t In this paper “cruciforms” are defined as any 4stranded cruciform (cross-shaped) duplex DNA structure, while the term “Holliday junction” is reservedfor cruciforms formed in a region of homology between2 duplex DNA molecules. A “nick” is an interruption of the phosphodiester backbone positioned continuity

of a single strand of DNA. Two appropriately nicks result in a “cut”, which interrupts the of a duplex DNA molecule.

0022~28:36,100/010117-18

$03.00/O

first postulated by Holliday (1964) to explain certain features of fungal recombination but has subsequently been incorporated into many models of break-join recombination (Whitehouse, 1982). The “diagonal” cleavage of two of the strands in the Holliday junction, such as by nicks at A a,nd C in Figure I(a), will resolve the intermediate into two recombinant molecules (Fig. l(a)). A cut on the opposite diagonal (nicks at B and D in Fig l(a)) will return the chromosomes to their initial configurations except for a single-strand patch of exchanged material. Several enzymes have been isolated that cleave model Holhday junctions. Endonuclease VII of phage T4 (the gene 49 product) or endonuclease I of phage T7 (the gene 3 product) produce diagonal cuts on cruciform DNA in vitro (Mizuuchi et al., 1982; de Massy et al., 1987). Both enzymes can also cut branched (Y-shaped) duplex DNA molecules (Jensch & Kemper, 1986; de Massy et al., 1987); one of their functions in vivo may be to produce mature linear phage DNA from the highly branched replication intermediates found in the cell. An enzyme capable of cleaving cruciforms has also been purified from mitotically growing Saccharomyces cerevisiae cells (West & Kijrner, 1985; Symington & Kolodner, 1985). This latter activity does not cut’ branched 117 0

1990

Academic

Prrvs

Limited

118

A. F. Taylor and G. M. S’jmith

Nicks A and

(b)

at C

Nicks at B and D

BornHI

E-CO RI

BornHI

Figure 1. (a). Interaction of 2 double-stranded DNA molecules. via the swapping of single strands, to produce a “Holliday” recombination intermediate. The isomerization of the structure to an open cross shape is shown (Potter & Dressler, 1978). The resolution of the intermediate into molecules parental or recombinant for flanking markers is shown. (b) The frozen cruciform used in these experiments, showing the sizes of the 4 arms, and the convention used for identifying strand interruptions. The restriction enzyme “sticky ends” on the end of each arm are marked. The star shows the position of the 32P label used in these experiments. The strands identified by the letters A, B, C and D are from cd’, c&L, at@ and attR, respectively. The sizes of the arms are from the nucleotide sequences of attP (Landy & Ross, 1977; Sanger et al., 1982) and of attB (McKittrick, N. H., Taylor, A. F.. Bauer. C., Gardner, J. & Abcarian. P., unpublished results). The arrows labeled A to D indicate positionsat the base of the cruciform at which nicking might occur,

and are referred to throughout the paper.

DNA (Evans & Kolodner, 1988), but there is at present no genetic evidence for the enzyme’s involvement in recombination. A Holliday junction joining two circular DNA molecules produces a characteristic “figure 8” structure. Such structures have been seen in several phage and plasmid DNAs extracted from E. coli, and in several cases are demonstrably joined in regions of homology (Thompson et al., 1975; Potter & Dressler, 1976; Valenzuela & Inman, 1975). In

two casest,he Holliday junctions so ident)ified have been shown to be recombination intermediates (Benbow et al.. 1975; Ikeda & Kobayashi, 1978). However, none of t’he Holliday junctions yet observed in E. coli can be definitelv attributed to t,he RecBCD pathway of recombina,tion. Physical and genetic characterization of’ the RecBCD pathway has been great,ly facilitated bj the use of mutants of phage 2 that can recombine only via that, pathway. Experiments with 2 showed that recombination by the RecBCD pat,hwa,y can proceed with very limit,ed DNA synthesis (Stahl ef nl., 1974). implying a brea,k-join mechanism of recombination. The further observations t,hat hetero-duplex D?iA accompanies recombination by thtk RecBCD pathway (Stahl et nl., 1974) and t,hat this recombination appears to be reciprocal (Sarthy & Meselson, 1976: Stahl et al.. 1982) narrowed the possible mechanisms of recombination and suggested a Holliday junction might) be an intrrmediate. lnitial observations t’hat) Chi-stimulated recombination. which is RecBCDdependent appeared non-reciprocal were subsequently shown to derive from t’he packaging mechanism of phagr j. and not, from an intrinsic property of the recombination mechanism (Kobayashi et al.. 1984). Gene products known to be involved in the RecBCD pathway of recombination include the RecA protein, RecBCD enzyme, DXA polymerase 1. DNA gyrase. DNA ligasc and t,he E. coli single-stranded DNA binding protein (for a review. see Smith. 1988). Purified RecA protein. DNA polymerase I and E. co& DNiZ liga.se have been used to prepare Holliday junct,ions in r+t~o and hence (West,

must al..

not, be sufficient

for

t,hrir

resolutjion

1983). Purified DNA gyrase can break phosphodiester bonds (Gellert, 1981) but does not, cleave cruciforms in z&o (Mizuuchi et al., 1982). RecBCD enzyme, which can also cleave phosphodiester bonds (for a review. see Taylor. 1988) k t,herefore. by elimination, a good candidate for the enzyme that resolves Holliday junct’ions, as has been proposed, for example, by Faulds et al. (1979). Genetic evidence demonstrates the requirement for RecBCD enzyme in recombination in E. ~01% but it is not known with certainty which of its ma,n> activities are required for recombination. RecBCD enzyme is needed for the RecA-dependent generalized recombina,tion of newly introduced TINAs wit,h the chromosome or with each other (for a review, see Smith, 1988). Such DNAs include chromosomal l>NAs mobilized by t)he F-factor and chromosomal fragments packaged in t,ransducing phage. Recombination of established plasmids, F-factors or bacterial chromosomeswith themselves or ea,chother is typically not RecBCD-mediated. apparently due to the lack of duplex DNA ends needed for the entry of RecBCD enzyme (Taylor Cyr, Smith, 1985). RecBCD enzyme is a large multifunctional nuclease with ATP-dependent double and single-sbrand exonu(‘leaseactivities and an ATP-stimulated single-strand endonuclease activit,y (for a review, see Taylor, 1988). Jt can unwind DNA, either transiently or et

Action of RecBCD

Enzyme

permanently, as it moves unidirectionally along duplex DNA (Taylor & Smith, 1980). The enzyme cannot endonucleolytically cleave duplex circular DNA but can cut single strands of duplex DNA while it is unwinding them. Such cutting can be sitespecific, at Chi recombination hotspots, or can be seemingly non-specific, as occurs during the degradation of duplex DNA (Ponticelli et al., 1985; Taylor et al., 1985). In summary, its role in recombination, its plethora of nuclease activities and its ability to move through duplex DNA make it plausible that RecBCD enzyme may resolve Holliday junctions.

2. Materials

and Methods

on

Cruciform

119

DNA

from the A,,, of the solutions, assuming 40 pg/ml per A 260 unit for the partially double-stranded oligonucleotides. Phosphates were added to the 5’ ends of the oligonucleotides by incubating 2.5 nmol of oligonucleotide with 25 nmol of ATP and 10 units of polynucleotide kinase for 30 min at 37 “C in linker-kinase buffer (Maniatis et al., 1982). A small amount of [Y-~~P]ATP was included, and the concentration of oligonucleotide was estimated from the trichloroacetic acid insoluble radioactivity incorporated, assuming 100% phosphorplation. Concentrations so estimated were typically about 75”/, of those estimated by A260. The EcoRI oligonucleotide cap was labeled at its 5’ end by incubating 5 pmol of the oligonucleotide cap with 17 pm01 of [y-32P]ATP (New England Nuclear, 3000 Ci/mmol) with 2 units of polynucleotide kinase for 30 min at 37°C in 14 ~1 of linker-kinase buffer (Maniatis et al., 1982).

(a) Enzymes

Restriction enzymes,polynucleotidekinaseandphageT4 DPU’A ligase were obtained from Bethesda Research Laboratories or New England Biolabs and used as described here or by Maniatis et al. (1982). RecBCD enzyme was purified as described (Amundsen et al., 1986). Fraction IV of the enzyme was used for all of the experiments shown in the Figures. RecBCD enzyme units are those of Eichler & Lehman (1977). Fraction IV had a specific activity of 3.6 x lo4 units/mg protein. Fraction V, produced from fraction IV by heparin-agarose chromatography, is virtually pure, and has a specific activity of 3 x lo5 units/mg (Amundsen et al., 1986). Fraction VI was prepared from fraction V by glycerol gradient centrifugation and lacks the minor contaminant bands present in fraction V (A. F. Taylor, unbublished results). The specific activity of fraction V, the known molecular weights of the subunits and the assumption that the active enzyme contains one copy of each subunit, yield the estimate of 6 x lo9 enzyme molecules per enzyme unit. (b) Preparation

of substrates

(i) Preparation of cruciform DNA Ml3 clones containing the bacterial

and phage 1 attach-

ment siteswere a gift from Bernard de Massy and Robert Weisberg (de Massy et al., 1987). Clone mLD5 carries attP, mLD12 carries attL, mLD14 (erroneously called mLD4 in the original publication) carries at@ and mLD16 carries attR. Virus was grown in E. coli strain S1386 [del(lac-pro) hsdS supE thi 1amB rpsL (P’Zac-pro lacIQ)] from Stanley Brown, Hutchinson Cancer Research Center. Ml3 phage was purified by precipitation with polyethylene glycol, followed in some cases by CsCl density gradient centrifugation. The cruciform DNA was prepared by annealing Ml3 DNAs containing complementary insert sequences, followed by restriction enzyme digestion of the restriction sites at the ends of the inserts (de Massy et al., 1987) and purification on a nitrocellulose column (Evans & Kolodner 1988). Cruciform DNA made from phage that

had not been CsCl-purified was further purified on a preparative

(iii) Capping of cruciforms Cruciform DNA (3 nM in molecules) was incubated with BamHI oligonucleotide cap (1 PM) and Hind111 oligonucleotide cap (50 nM) in 50 ~1 of linker-ligation buffer containing 100 pg bovine serum albumin/ml (Maniatis et al., 1982); @5 unit of T4 DNA ligase was added and the mixture incubated overnight at 15°C. The high concentration of BamHI oligonucleotide cap was designed to compete with the intramolecular ligation of the 2 BamHI sites in the cruciform. Following heat inactivation of the ligase, NaCl was added to 100 mM, EcoRI restriction enzyme added and the mixture incubated until all of the cruciforms, which had dimerized at their EcoRI sites, had returned to monomer size. DNAs were then purified by extractions with phenol and chloroform, followed by precipitation with ethanol. Labeled EcoRI oligonucleotide caps were added in either of 2 ways. In some experiments they were ligated onto the a-capped cruciform described above. In other experiments the 32P-labeled EcoRI oligonucleotide cap was included (at 20 nM) in the ligation reaction described above, and the subsequent treatment with EcoRI enzyme omitted.

agarose gel.

(ii) Preparation of oligonucleotide caps Oligonucleotides were synthesized on an Applied Biosystems model 380B DNA synthesizer and were precipitated with ethanol twice before use. Their sequences, presumed secondary structure and relevant restriction sites are shown in Fig. 6. Concentrations were estimated

(iv) Labeling of cruciforms Cruciform DNA (either uncapped or with BamHI and Hind111 caps added) was uniquely labeled at the 3’ end of the EcoRI site by incubation with appropriate deoxy- and dideoxynucleoside triphosphates and the Klenow fragment of DNA polymerase I (de Massy et al., 1987): 70 fmol of cruciform was incubated with 100 pmol of dATP, 1 nmol of dideoxy GTP and 3 units of the Klenow fragment of DNA polymerase I for 10 min at 20°C in 50 ~1 of “medium salt” restriction enzyme buffer (Maniatis et al., 1982). Then 2.5 pmol of [cr-32PJdTTP (New

England Nuclear, 800 Ci/mmol) was added and incubation continued for 2 min. The labeled DNA was purified by extraction with phenol and chloroform, recovered by precipitation with ethanol and dissolved in 10 miw-Tris’ HCl (pH %O), 1 mM-EDTA. Concentrations of cruciforms were estimated by precipitation of samples with trichloroacetic acid at appropriate stages.

(c) RecBCD enzyme reactions with cruciform

substrates

RecBCD enzyme reactions were carried out 100 mm-Nacl, 50 mM-3-[N-morpholino]propanesulfonic

in

A. F. Taylor and c71.R. Smith

120

acid-NaOH (pH 7.0). 5 mM-ATP. 1 m&I-MgCl,. 1 mM-CaCI,, 1 mM-dithiothreitol. Reactions were typically in 10 ~1 and were stopped by addition of 0.2 vol. 5004 (w/v) sucrose, @2% (w/v) bromphenol blue, 2% (w/v) xylene cyanole FF. 50 mM-EDTA. 0.5% (w/v) sodium dodecyl sulfate. Samples were typically heated to 70°C for 5 min before electrophoresis. or boiled for 2 min for denaburation.

(d) Gel electroph,oresis (i) -C’atice gels Agarose (Sigma type I) gels (100 ml. 16 or 20-place) were poured in 21.5 cm x 12.7 cm trays (Ellard Instrumentation. Seattle. WA) in THE buffer (Maniat,is et al.. 1982). Gels were typically run for 16 h at 1.2 V/cm. dried onto \Vhatman DE51 paper under vacuum at 80°C and autoradiographed at -70°C with Du Pont Cronex Lightning Plus intensifying smwm

(ii)

Two-dimensionnl

gels

Agarose gels (15 cm long, 100 ml. 1.40/0 (w/v)) in TBE buffer were cast in 12.7 cm wide trays (Ellard Instrument,ation. Seattle. WA). The layout of the gel and the manipulations involved in runmng it are shown in Fig. 3. A comb was pla,ced. in the normal orientation, 2.5 cm from the start of the gel. It had several 0.3 cm x 0.1 rrn teeth on one edge for samples run only in the 1st dimension (1st dimension samples) and a single tooth (01 cm x @l cm) for the sample being analyzed (t,he 2dimensional sample). In line with this tooth. but at the far end of the gel. a 2-tooth comb was placed. at right-angles t,o the 1st dimension. for the samples that ran only in the denaturing dimension (2nd dimension samples). After reaction with RecBCD enzyme the labeled cruciform DNA was extracted first with phenol, then with chloroform. and precipitabed with ethanol with tRNA as a carrier. Part, of the redissolved reaction mix was loaded into the 2-dimensional sample well. Another fraction of the reaction mix. and size standards, were loaded as 1st dimension samples. and the lst, (native) dimension of the gel was run. Electrophoresis conditions are given in the Figure legends. The region behind the comb was cut off and discarded. The gel was then soaked in several changes of 50 m&r-NaOH. 1 mM-EDTA for 3 h. The section of gel containing the 1st dimension samples was excised and stored in the NaOH/EDTA solution. The remainder of the gel was t,urned at right-angles. returned to the gel box. and submerged in the NaOH/EDTA solution. The 2nd dimension samples (size markers and a sample of the reacted cruciform) were added, and the 2nd (denatured) dimension of the gel was run at 4°C with buffer recirculation. The excised section and the gel were then neutralized by soaking for 1 h in 1 M-Tris.HCl (pH 7.6), the excised se&ion returned to its original position and the gel dried and autoradiographed as above.

(ii) KpnZ digest of cruciform Labeled cruciform was digested salt” buffer (Maniatis et al.. 1982).

(e) Size

staw&rds

KpnI

in “low

(iii) IV! 13 mLDl2 and mLDl4 inserts Replicative forms of the plasmids were made from “minipreps” (Maniatis et al., 1982) of phage-infected cells. The inserts were excised from the vectors, and labeled at, t,heir BcoRI ends by incubation with unlabeled dATP. [a-32P]dTTP and the Klenow fragment of DNA polymerase I. The inserts were purified by agarose gel rlectrophoresis. (iv) BstElI digest of lnmbdn i, cl857 sus~\7 x * A DNA was digest,ed in “medium salt,” restriction enzyme buffer (Maniatis et al.. 1982) with 1ZstEII. and the 3’ ends of the fragmentas were labeled by incubation with the Klenow fragment of l)?iA polymerase I in the presence of unlabeled dGTP and [a-32P]dTTP. Fragment sizes are 8.45. 7.24. 637, 5.59. 4.82. 1.32. 3.68, 2.32, 1.93. 1.37. 1.26. 0.7, 02 and @I kbt (Sanger et al.. 1982). (v)

Single-strand

size murkers

Cruciform DNA was labeled at all of its 3’ termini 1)~ incubation with the Klenow fragment of DNA polymerasr 1 in the presence of unlabeled dATP and dGTP, and [a-32P]dTTP and [c(-32P]dCTP. Samples were denatured by boiling before loading. Strand sizes are 1.7. 1.2. I.0 and 0.5 kb (Sanger et al., 1982: McKittrick. N. H.. Taylor. A. F.. Bauer. C.. Gardner. J. & Abcarian. P.. unpublished results).

(f) Action

coli e&acts

of E.

on capped

wuciforms

(i) Preparation and reaction of E:. coli c~&mts Cultures (25 ml) were grown and cell ext’rauts prepared as described (Ponticelli et al.. 1985). The extracts were not dialyzed and were used immediately after their protein concentration had been measured by the method of Bradford (1976). Typical reactions in 10 ~1, cont’ained 50 m,n-3-[A-morpholinolpropanesulfonic 25 mM-NaCl, 5 mM-ATP. 10 rnM-MgCl,, (pH 7.0). acid-NaOH 1 mM-dithiothreitol and 10 pg extract protein. t,ogether with 1 fmol of fully capped cruciform DNA with a 321’ label on the 5’.phosphate of the EcoRT oligonucleotide cap. Aft,er incubation (typically 2 to 10 min at 37°C’). reaction was stopped by addition of 0.2 vol. stop mixture (section (c). above) containing 100 rnx-EDTA.

(ii) Genotypes of E. coli strains examined Strain V414: sbcBl5 recB21 rrcC22 zthA1

endA pheSl1

lew6

(strain BW9061 of B. Weiss, ,Johns Hopkins Medical School). Strain S785: recA trpR(?) met hsdS .supE supF (from K. Sprague. CTniversity of Oregon. Eugene). St,rain VH4: recB rec(l h,sdS (from N. Murray. Ilniversity of Edinburgh). argE3

(i) Partial Hinfr digest of cruciform Cruciform (@X fmol 32P-labeled at the 3’ end of the EcoRI site) was mixed with 1 pg of sonicated calf thymus DNA, in 50 ~1 of 50 mr/r-Tris.HCl (pH 8.0) 10 mM-MgCl,, 50 mM-NaCl, and digested with 0.2 unit of HinfI for 2.5 min at 37°C.

with

his-4

supE44

t Abbreviations bp. base-pair(s).

thi-1

mtl-1

used: kb. lo3

thr-7

bases

or base-pairs;

Action of RecBCD

Enzyme on Cruciform

Strain V830: recB21 recC22 sbcB15 h&R his-4 thr-12 leu6 thi-1 proA argE3 thr : : TnlO lac Yl galK2 ara-14 xyl-5 mtl-1 rpsL31 tsx-33 (strain CES200 of C. Shurvinton, University of Oregon, Eugene).

3. Results (a) Action of RecBCD enzyme on palindrome-derived cruciforms

Duplex DNA molecules containing inverted repeat sequences (“palindromes”) may adopt a cruciform structure in which each inverted repeat is base-paired with its complement on the same strand of DNA. The topology of the junction region of such a molecule is identical with that of the Holliday junction. The linear duplex form of the molecule is energetically favored, but in circular molecules the cruciform state may be induced by DNA supercoiling (Gellert et al., 1978). Such cruciforms were used as substrates for the first Holliday junction resolving enzyme discovered, endonuclease VII of phage T4, which converts the circular substrate to a linear duplex by nicking opposite strands at the base of the cruciform (Mizuuchi et al., 1982). A cruciform extruded from the same supercoiled plasmid was, however, resistant to the action of RecBCD enzyme (M. O’Dea, K. Mizuuchi & M. Gellert, personal communication). We show below (section (d)) that a different type of cruciform is similarly resistant’ to RecBCD enzyme if it is similarly devoid of duplex ends. Removal of the tips from extruded cruciforms, to produce nearly-flush termini, would allow the entry of RecBCD enzyme into the cruciform-containing DNA molecules (Taylor & Smith, 1985). Reaction of such substrates with RecBCD enzyme did reveal the presence of specific cuts, but these cuts were not at the same positions as those produced by T4 endonuclease VII (M. O’Dea, K. Mizuuchi & M. Gellert, personal communication). RecBCD enzyme unwinds and rewinds DNA, producing travelling loops of single-stranded DNA in its vicinity as it travels through duplex DNA (Taylor & Smith, 1980). Inverted repeat regions in a long duplex DNA molecule have the potential to undergo intra-strand base-pairing during such unwinding by RecBCD enzyme, hence producing cruciforms in linear duplex DNA. When such DNA, containing very long (2 x 42 kb; Shurvinton et al., 1987) palindromes, was reacted with RecBCD enzyme, cruciforms with arms up to the size expected were frequently seenin the electron microscope (Taylor, A. F., Shurvinton, C. E., Stahl, F. W. & Smith, G. R., unpublished results). Examination of such reaction products by gel electrophoresis showed enzyme-dependent double-strand cuts whose positions were consistent with RecBCD enzyme cleavage of the cruciform. The large size of the inverted repeats made the location of the strand interruptions impossible. Similar experiments with duplex DNA carrying a shorter palindrome (2 x 265 bp; Leach &, Stahl,

DNA

121

1983) showed similar cutting of the DNA into two unique-sized fragments. The location of one of the two strand interruptions necessary to cleave the presumed cruciform was studied: it could lie anywhere between the tip and the base of the fully extruded cruciform (Taylor, A. F., Shurvinton, C. E., Stahl, F. W. & Smith, G. R., unpublished results). The travel of a RecBCD enzyme molecule through the DNA presumably allows extrusion of the cruciform, followed by cleavage of the cruciform by one or more RecBCD enzyme molecules. As the extruded palindrome can isomerize back t’o the fully duplex state, we could not tell whether the spectrum of nicks observed resulted from the enzyme always nicking at the base of the cruciform (with cruciforms extruded to different extents) or whether the enzyme can nick at various places along the extruded cruciform arms. (b) RecBCD enzyme cuts “frozen,” open-endedcruciforms The problem of uncertainty in the position of the base of the cruciform was circumvented by the use of a substrate whose ability to branch migrate was severely limited (a “frozen” cruciform). This substrate, constructed from DNA containing the phage lambda attachment sites propagated in phage M13, has four non-homologous arms (de Massy et al., 1987) joined at the homologous 15 base “core” of the attachment site (Fig. 1(b)). The structure thus has the topology of a true Holliday junction but differs from it in not having two pairs of homologous duplex arms. The cruciform was labeled with 32P at the 3’ end of the EcoRI site (Fig. l(b)): comparison of lanes 9 and 13 of Figure 2 shows that essentially all of the label was, as expected, in the longest strand of the cruciform. Reaction of the labeled cruciform with RecBCD enzyme (lanes 4 and 5) produced four major bands, in addition to some residual substrate band. The strongest product band ran slightly faster than a double-stranded DNA size-marker (lane 1) made from the replicative form of the Ml3 derivative that supplied the 1.2 kb strand of the cruciform. As shown in Figure 1, cleavage at A and C would produce a duplex DNA of that length with a single nick in it. The absence of the nick from the size-marker may explain its slightly slower mobility. The next most prominent band produced by RecBCD enzyme (lane 4) migrated slightly slower than the marker in lane 2, which shows the expected position for nicks at B and D (Fig. 1): again the presence of a nick in the labeled reaction product may explain the slight difference in migration. The nature of the two faint,er bands (above and below the BD cut band) is not clear: the slower migrating band may arise from nicks at A and B or A and D, while the faster one may represent nicks at C and D. These faint bands were not always seen (see Figs 7 or 8) and have not been investigated further. Similar minor products are also seen with T7 endonuclease I (de Massy et al., 1987).

122

A. 3’. Taylor

and 0. R. Smith

EDcut

uncut strand

ACcut

NC nick

I

2

3

4

5

1

6

7

8

3

IO

II

12

13

Figure 2. Cleavage of open-ended cruciforms by RecBCD enzyme: 625 fmol samples of cruciform! uniquely labeled at, the 3’ end of the EcoRI end, were reacted with 63 unit of RecBCD enzyme for 5 min at 37°C (approx. 10 enzyme molecules/cruciform molecule). The sample in lane 4 was not heated, that in lane 5 was heated at 70°C for 5 min before loading, while that in lane 10 (as well as the markers in lanes 11 to 13) was boiled for 2 min before loading. Lanes 3 and 9 contain native and boiled unreacted cruciform DNA, respectively, while lane 13 contains unreacted cruciform labeled on all 4 strands and boiled before loading. Lanes 1 and 2 are full length, un-nicked, duplex molecules of the size and sequence that would be produced by either diagonal cleavage of the cruciform. Samples were analyzed on a 2% (w/v) agarose gel in TBE buffer. The cruciform is designated X in the Figure, The legends BD cut, AC cut and C nick refer to resolutions of the cruciform by nicking at the sites so labeled in Fig. l(b). The cartoon below the gel shows (not to scale) the positions of relevant size markers generated by cutting at the unique KpnI site on the cruciform (lanes 7 and 12) or by a partial HinfI digest of the cruciform (lanes 8 and 11). Only the Hi&I sites in the right arm of the cruciform are shown. Partial digestion with Hid produces labeled strands of length 98, 611, 693, 770 and 932 nucleotides while digestion with KpnI produces a strand of 783 nucleotides. Nucleotide 1 of the core region of the attachment site, as conventionally numbered (Landy & Ross, 1977) is 953 nucleotides from the label. The well separated double-stranded B&E11 size markers in lane 6 are 2.32, 1.93, 1.37, 1.26 and 070 kb long. The marks on the ends of strands denote 3’ ends, and the star marks the 32P label, as in Fig. 1.

Action of RecBCD

Enzyme on Cruciform

A marker for the product of a cleavage at B and C was made by a partial Hinf’I digest of the [32P]EcoRI end-labeled cruciform (lane 8): the nearest Hi&l site is 23 base-pairs towards the 32P label from the base of the cruciform (Landy & Ross, 1977). The appropriate band was identified by reference to the unique KpnI site 170 bp from the base of the cruciform (lane 7). After reaction with RecBCD enzyme (lanes 4 and 5) there was a smear extending from the position of the AC cleavage downwards and ceasing abruptly at the position expected (lane 8) for duplex DNA nicked at B and (1. The most probahle explanation is that the nicks at, A and C were made first, producing a 1.2 kb duplex DNA with a nick opposite B and that subsequent enzyme action converted this DNA into a set of molecules with nicks at various positions between the upper BarnHI site and A or B. In other experiments (Figs 4 and 5 and results not shown) a distinct’ band at the position of a BC cleavage was seen. The sa,mples shown in the right half of Figure 2 were denatured before electrophoresis, to identify the position of the strand interruption on the 32P-labeled strand of the cruciform. As described above, a partial Hinff digest of the labeled cruciform identified the position expected for nicking at C. at the base of the cruciform. The sample denatured after RecKCD enzyme reaction (lane 10) some full-length labeled strands (as showed expected for unreacted molecules or those nicked at B and 1)). The bulk of the labeled material, however, ran as a doublet at and near the position expected for a nick at C. as identified by the partial HinfI digest product,s of the labeled cruciform (lane 11). The source of the doublet is not known, t’hough it was also seen in one of t,he two-dimensional gels described below. The major band produced by the action of RecBCD enzyme on the labeled cruciform was thus shown, on the non-denatured portion of Figure 2, to have the size expected from nicks at A and C, assuming t’hat the reaction product was fully duplex. Support for that assumption comes from the denatured samples in Figure 2; the major reaction product did indeed result from a nick at or near C. This argument, however, is based on the assumption that the material in the major band in the denatured sample (that nicked near C seen in lane 10) came from the major band in lane 4, that tentatively identified as a duplex molecule produced by nicks at A and C. The results shown in Figure 2 were obtained using fraction IV of RecBCD enzyme, whose specific activity is 5 10 y/, of that of nearly homogenous enzyme (fraction V) but which has no detectable ATP-independent double-strand exonuclease activity. RecBCD enzyme-dependent reaction products identical with those seen in Figure 2, with or without denaturation, were observed when more highly purified RecBCD enzyme was used. Either fraction V, which has barely detectable levels of contaminating polypeptides, or fraction VI, whose only detectable

123

DNA

polypeptides are the RecB, RecC and RecD polypeptides, gave products indistinguishable from those obtained with fraction IV (data not shown). The cleavages observed must therefore be a result of the action of RecBCD enzyme and not of a contaminant in the preparation. Fraction IV was used for the remainder of the results reported here. In the experiment shown in Figure 2 there were an estimated 2.5 enzyme molecules per duplex DNA end, as calculated from the specific activity of nearly homogenous enzyme (see Materials and Methods). Similar reaction products were also seen when there was an excess of substrate over enzyme as seen in the right half of Figure 7, where the 0.07 units and 0.22 units of enzyme used correspond to 0.2 and 0.6 enzyme molecules per duplrx end. (c) Two-dimensional

gel analysis

of reaction products

A two-dimensional gel analysis. using native and denaturing agarose gel dimensions, was used to confirm and extend the conclusions from the previous section. As detailed in Materials and Methods and illustrat’ed in Figure 3, the reacted material for the twodimensional analysis was first run on a non-denaturing (native) slab gel together with another portion of the reaction material and size standards. The gel was then denatured in alkali and the portion of the gel containing the size standards and reaction sample was excised and put aside. After being run in the second denaturing dimension (with size standards added) the gel, together with the excised portion, was neutralized, dried and autoradiographed. As can be seen in Figure 4(c). the spots in the two-dimensional gel were not in perfect, register with the corresponding bands of a non-denatured reaction sample run in the first dimension of the gel. The spots can be identified by comparing t’he mobilities of the non-denatured bands with those of the non-denatured size standards and then comparing them to Figure 2. There were three main bands in the RecBCD enzyme reaction products run in the native dimension; their tentative identification as full-length cruciform, cruciform nicked at bot,h A and C, and cruciform nicked at both B and C, was confirmed by comparing the mobilities of the spots in the denaturing dimension with that of denatured size standards. In the denaturing dimension all the material migrated either as full-length material or as material nicked at C. Most of the material that ran as full-length cruciform in the native dimension also did so in the denaturing dimension though some of it ran as fulllength cruciform that was nicked at C. The appearance of the nicked cruciform was investigated in a time-course of RecBCD enzyme reaction with cruciforms (Fig. 4). Unreacted cruciform (Fig. 4(a)) ran as a tight spot in the native dimension but showed a small amount of apparently random nicking in the second dimension. This sample, unlike that in Figure 4(b) and (c), was stored overnight as a pre-

124

A. F. Taylor and G. R. Smith

W?-lple @fl;nelecE%:res2 E, =~~” dimension samples

sample / (1) Denature gel (2) Excise 1st. dim. (3) Rotate gel

*(

/ / (1) Load 2nd. dim. samples

v

Reaction sample Size markers

CD(2) Run denatured dimension

dim. samDles (2) Dry:nd autoradiograph I

1

Native electrophoresis Figure 3. Diagram of the 2-dimensional excised and discarded before denaturation. the 2nd dimension slots was also discarded region bounded by the 2 sets of slots.

gel procedure. In practire, the area of gel behind the 1st dimension slots was It is shown here to aid visualization of the manipulations. The area behind after drying: the area of gel shown on the autoradiographs corresponds to the

cipitate, and presumably suffered radiation damage. After a one minute reaction with RecRCD enzyme (Fig. 4(b), most of the cruciform was still intact, but there was a distinct spot identified (as described above) as a cruciform nicked at C. After a ten minute reaction Fig. 4(c), predominantly doublestrand cut material was seen. Reaction of RecBCD enzyme with this “frozen” cruciform initially produced a nick at C and only later a double-strand cut at A and C. Nicks at A would not be detected: the strand was unlabeled in

these experiments. Cleavage of this cruciform by RecBCD enzyme probably resulted from independent nicks at, A and C. The samples analyzed in Figure 4 were all from a single time-course. An intermediate time-point (5 min) from a separate reaction is shown in Figure 5. As might be expected, this reaction produced about equal amounts of nicked and double-strand cut material. Figure 5, like Figure 2, shows the reaction products to be doublets in the second dimension. All the reaction products, which

3.7

lmin

Sample

2.3

I.9

of reaction

Standards

I.4 I.3

Dimension

0.7

llx

I IZ

I g

I

I l,l$ 1: IZly I c Ito IWl~ ;;;e

I

I I I

1 I I

Figure 4. Time-course of reaction of KecBC~D enzyme with open-ended final volume of 150 ~1 with 1.2 fmol of EcoRI (3’-‘2P)-labeled cruciform EDTA to 10 mM. 1 pg of tRNA and sodium acetate to @3 M. After 1 TE: 2 ~1 of tracking dyes and sucrose were added. and 0 ~1 was added denaturation of the gel the remainder of the reaction was added to the dimension6 (and their sizes, in kb, marked on the edges of the panels) Methods. The 1st dimension was run at 2.7 V/cm for 7 h and the 2nd sites so labeled in Fig. 1 (b). The diagram shows t,he directions of na.tive reaction products in the 2 dimensions.

nlrked

(b)

t

---------em-----------

Size

----------------,-,----~

Native

nicked

( c )

IO min

I 3-7

II 2.3

l-9

II

BC cut

1.4 I.3

AC cut

0.7

I

cruriforms analyzed by I-dimensional electrophorexis. RecBCD enzyme reactions were carried out in a and 4.5 units of RecB
I

A. 1‘. Taylor

126

-Native

dimension

and G. R. Nmith

__j

I.92

x

I.37 I.26

Nit :ks at C

AC CUi

BC cut

Figure 5. High resolution Y-dimensional gel. A RecB(‘D enzyme reaction was carried out (for .? min) and processed. ar described in the legend to Fig. 3, with 5 fmol of cruciform and 6 units of Re(~BCI> enzyme in a volume of 200 ~1: 70”,;, of the sample was run as the Z-dimensional sample and 20~~~ as the 2nd dimension sample. together with a labeled BstEIl digest of /1 (whose sizes in kb are marked on the Figure). The 1st dimension was run for 13 h at 1.7 V!cm and t’he 2nd dimension for 22 h at 1.4 V/cm. AC cut. RC cut and nicks at C refer to the cut-sit’es so labeled in Fig. l(b).

resolved in the native dimension as separate species. clearly arose from two closely spaced nicks near C. the base of the cruciform. Figure 2 shows that one of the nicks was extremely close to the base of the cruciform and the other was about 100 nucleotides away. The two nicks may have arisen from R#ecBCD enzyme molecules travelling down different arms of the cruciform and then nicking near its base.

were

(d) RecBCD

enzyme does not cut cruciforms ends capped

with all

The observation that the cleavage of cruciforms by RecBCD enzyme occurred as two separate nicks raised t,he possibility that the two nicks might be effected by separate enzyme molecules travelling down separate arms of the cruciform. We tested this idea, and checked whether the RecBCD enzyme attacked cruciforms via their ends, by blocking the ends of the cruciform with synthetic oligonucleotides. A series of oligonucleotides was synthesized. shown in Figure 6, which can base-pair to form a “hairpin” shape and a restriction enzyme sit’e. enabling them to be ligated onto the termini of the

frozen cruciform (Fig. l(b)). When ligat,ed ont,o the ends of linear duplex DNA? these oligonucleotjides (referred to here as oligonucleotide caps, hut, colloquially named Taylomeres) greatly decreased the rate of degradation of the DNA by RecBCD enzyme (data not shown). Similar hairpin structures, the ends of vaccinia. virus DXA, inhibit the double-strand exonuclease activity of Hemophilus in$uenzae exonuclease V (Orlosky & Smith, 1976). Frozen cruciforms were prepared with caps on all four ends. The EcoRI caps used were 32P-labeled at t’heir #ends. hence putting a unique 32P label near the 3’ end of the EcoR.T site of the (aruciform.

as

in

the previous experiments. Capped cruciforms were reacted with RecBCD enzyme, and t.he caps (but’ not the 32P label) removed before gel electrophoresis, to allow the detection of either a double-&and cut at A and (1 or a nick at C. If all the oligonucleotide caps remained attached, the cruciform would reanneal after denaturation even if it were nicked. As can be seen in Figure 7. RecBCD enzyme made neither

nicks

nor double-strand

cuts

on czruciforms

without open ends (lanes 4 to 7). AR a control for this experiment, the oligonucleotide caps were removed before RecBCD enzyme reaction, by diges-

Action (a)

HlndIU

sf RecBCD Enzyme on Cruc

"Taylomere"

HindID 5G&TCGAGGGCCTA

AGAGCTCCCGCA I I x7701

(b)

BarnHI

"Taylomere"

Bun7 HI S-%TCGAGGGCCTA GAGCTCCCGGA I I XI-l01

(c)

,!?coRI

"Taylomere"

EcoRI

d=GATCGATGGCCTA GCTAGCTACCGGA I I C/al sequences and predicted Figure 6. Nucleotide secondary structures of the 3 self-complementary oligonucleotides (oligonucleotide caps) used to block the ends of the cruciform. The “sticky” ends used to ligate the oligonucleotides to the cruciform are marked, as are the restriction sites used to remove the caps from the capped

cruciform. tion with CEaI, BamHI and HindIII; the right half of Figure 7 shows that the normal spectrum of nicks and cuts was made (lanes 10 to 13). Thus RecBCD enzyme needsaccessto at least one duplex DNA end for it to cleave cruciforms. The experiments also confirm that oligonucleotide caps do indeed block the entry of RecBCD enzyme into duplex DKA. (e) RecBCD enzyme nicks cruciforms with one open end To determine whether the enzyme needs accessto more than one end in order to cut the cruciforms, reactions were carried out with various cruciform termini capped. The results of such experiments, again with a unique 32P label on the 3’ end of the EcoRI site, are shown in Figure 8. The caps were not removed before denaturation: this allowed estimates of the efficiency of ligation of the oligonucleotide caps onto the ends of the cruciforms. The control for these experiments was the set of reactions, shown in lanes 7 to 9, in which the cruciform was never capped and was not treated with

127

Hind111 or BamHI. Virtually identical results were seenwith substrates that had been capped and then uncapped (with BamHI and HindIII) or with uncapped cruciforms that had been treated with BamHI and Hind111 (data not shown). In the native portion of the gel, bands identified as the products of AC and BD cuts were seen(lanes 7 to 9). As is typically seen, the BD cleavage appeared before the AC cleavage but never approached it in intensity. The appearance of the nick at C, in the denatured part of the gel, paralleled that of the AC cleavage, as expected. When all the termini of the cruciform except for that at the labeled EcoRI end were capped, no double-strand cutting was seen (lanes 1 to 4, native samples), but the nick at C was still made with high efficiency (lanes 1 to 4, denatured). When the unreacted, three-capped cruciform was denatured and run (on a non-denaturing agarose gel: Fig. 8, lane 1) most of the label ran in the position expected for a native cruciform structure. The denatured cruciform must have reannealed to it’s native conformation after boiling. The t’hree bands running faster than native cruciform are molecules in which one or more of the ligations needed t’o attach the oligonucleotide caps to the cruciform did not occur. The fastest of these bands ran with a single strand of labeled, uncapped cruciform: it represents failure of ligation at the “lower” BamHT sit)e (Fig. l(b)). The next fastest band comigrates with the denatured substrate in lanes 15 to 18 (that with its Hind111 cap removed): it results from failure of ligation at the Hind111 site. However, in all cases, only a small fraction of the ligations were unsuccessful. The small amount of AC cleavage seenin lane 4 of Figure 8 may represent reaction by enzymes entering both at the uncapped EcoRI end and at another uncapped end. Such uncapped ends could occur if the 3’ overhangs on some of the termini were damaged, preventing them from base-pairing with the oligonucleotide caps. Despite the lack of AC cutting with the three-capped substrate, there was a normal, though possibly delayed, amount of nicking at C. The uncapped control made by the addition and subsequent removal of caps showed a similar delay in nicking at C, compared to the never-capped control (data not shown). Excess caps present in these reactions may have temporarily sequestered the RecBCD enzyme. Oligonucleotide caps, even though they are very short duplexes, are substrates for the exonuclease activity of RecBCD enzyme (A.F.T., data not shown). Thus, RecBCD enzyme molecules entering solely from the EcoRI end of the cruciform can effect a nick at C, but cannot produce the double-strand cuts at A and C (or at B and D) necessary to resolve the cruciform. The simplest hypothesis for the double-strand cuts with all ends open is for RecBCD enzyme molecules to enter at the EcoRT and Hind111 ends, travel along the DNA until they encounter the cruciform, and then nick the strands

128

A. F. Taylor

Caps removed:

After

reaction

and G. R. Smith

Before

I

reactron

Boll:

Cruci form

Labeled cruciform orm

-

2,32-

1.37-

I .26-

ClBC C-CT

cut rick

0,70-

I2

34

567

8

9

IO

II

12

13

14

Figure 7. React,ion of fully capped cruciforms with RecBCD enzyme. Fully capped cruciform DNA. ‘21’-labeled at t,he 5’ end of the &oRT oligonucleotide cap [and thus at t,he 3’ end of the KcoRT site on the cruciform). was incubated without (lanes 2 to 7) or with HamHI. HindIIT and CEaT restriction enzymes (lanes 8 t,o 13) in medium salt, restriction buffer (Maniatis et al.. 1982) to remove all caps. The restricted or mock-restricted cruciforms were diluted into a reaction mixture whose final composition approximated that for the RecBCD enzyme reaction mix (Materials and Methods). Samples (1 fmol) of the cruciforms with or without caps were reacted with 0, CO7 or OS! unit of RecIKD enzyme for 10 min at 37°C. Reactions were stopped by addition of EDTA to 5 mM, ammonium acetate to 2 x and 50 ng of sonicated calf thymus DNu’B. Sfter extraction with phenol and chloroform, t’he DNA was precipit,ated with et,hanol, and redissolved in TE. All samples. bot,h the 4-capped and uncapped, were t.hen reacted in medium salt. buffer (Maniatis et al.. 1982) with XhoI and CZnT to remove the caps. After addition of EDTA. sucrose and tracking dyes, samples were, where indicated. denatured by boiling for 2 min. Native (lane 1) and denat,ured (lane 14) size standards were prepared as described in Materials and Methods. Markers ;1C cut and BC cut refer to native material. C nick to denatured material.

whose

3’ ends

they

had

entered,

as shown

in

Figure 8. This 3’.entering hypot,hesis was tested by a reaction of RecBCD enzyme with cruciforms whose EcoRT and Hind111 ends were open (Fig. 8, lanes 15 to 18) but whose BamHI ends were blocked. In that reaction there was indeed nicking at C and doublestrand cutting at A and C. However, the nicks at A and C were seenonly after ten minutes reaction and were accompanied by the production of a larger specieswhose size, if fully duplex, would be about 1.5 kb. Such a fragment may be produced by nicking at C and D but cannot be produced by the simple rule of cutting only those strands whose 3’ ends were entered.

(f) RecRCD two

enzyme cuts cruciforms or mow open ends

with

The 3’.entering hypothesis was also directly ruled out by results with cruciforms whose Hind111 ends were capped (Fig. 8, lanes 11 to 14). Nicks at C and double strand cuts at A and C were the only detectable actions of RecBCD enzyme on this substrate. Bs entry cannot occur from the Hind111 end: at least one of the nicks needed for the double-strand cut must arise from an enzyme entering at a BamHI end, travelling down to the cruciform base and then nicking the strand whose 5’ end it entered, as shown in Figure 8. Nicking on the 3’-entering strand by enzymes coming from the BamHI ends would not, be

Action of RecBCD

Enzyme on Cruciform

DNA

129

Time (min):

AC cut

I

2

3

4

5

6

7

8

9

IO

I I

I2

13

14

15

16

I?

18

19

Figure 8. Reaction of RecBCD enzyme with cruciforms with various ends capped. Three-capped (HwLHT and HindIII caps) cruciforms. labeled at the 3’ end of the EcoRI end, were reacted with BamHI or HindTTI and prepared for RecBCD enzyme reaction as in Fig. 7. Reactions were with 6.25 fmol of cruciform and 0.3 unit of RecBCD enzyme for 1, 3 or 16 min at 37°C. The control reactions shown (lanes 6 to 9) are with EcoRI labeled cruciform that had never been capped. As shown above the gels (the star indicates the position of the 32P label), the substrate in lanes 1 to 4 was capped at the BumHI and Hind111 ends, that in lanes 11 to 14 was capped only at the Hind111 end while that in lanes 15 to 18 was capped only at the RamHI ends. After RecBCD enzyme reaction and the addition of EDTA, sucrose and tracking dyes. the reaction products were divided into 2 fractions, 1 of which was denatured by boiling. The native and denatured samples were run in parallel on non-denaturing 1 yO (w / v ) a g arose gels in TBE buffer for 3.5 h at 3.5 V/cm. The labels to the left of the gels identify the prominent bands. The numbers to the left of the denatured samples identify the position of unreacted cruciforms with 4, 3,2 or 1 strands covalently joined to the 32P label. Pu’umbers above the upper gel indicate incubation times (for both gels) in minutes: the marker lanes are labeled M. Kative size markers are a HstEIT digest of 1 and denatured size markers are the 4 labeled strands of the cruciform (see Materials and Methods).

In a separate experiin this experiment. ment (not shown), with a cruciform labeled at the EcoRT end and capped at the EcoRI and Hind111 ends. a 1.7 kb double-strand fragment (corresponding to a BD cleavage) was seen, which apparently arose from enzymes that had entered at both

detected

BamHI ends and then both made nicks on their 3’entering strands. Thus, RecBCD enzyme molecule(s) approaching the cruciform from only one direction made a nick at or near the base of the cruciform. Double-strand cleavage requires the enzyme to approach the cruci-

130

A. F. Taylor

t EcoK

and G. R. Smith

- EcoK

-EcoK -

- RecA

-ECOK -ExoI

t F

- RecBCD

RecBCD

7me

2

A ii w

Crlm form

2.3

I.9 BD cut

I.4 I.3 AC cut

2

4

6

0

IO

12

14

16

Figure 9. Cutting of fully capped cruclforms by h’. coli extracts: 10 pg of extract protein was usedfor each reaction. except those in lanes 6, 10 and 14, which received 50 pg. Reactions, as described in Materials and Methods. were for the times indicated. The relevant genotypes of the strains from which the extracts were made are noted above the lanes. Strains were V414 (lanes 1 and 2), 5785 (lanes 3 to 6). V84 (lanes 7 t,o 10) and V830 (lanes 11 to 14). Reartion products were identified by their apparent sizes, using the double-strand DNA size markers in lanes 16, and are identified at t,hr left of t,he gel. The sizes of the markers are indicated in kb.

form from at least t’wo directions. Enzymes appear, in different experiments, to be able to nick at the base of the cruciform on either (but not both) of the strands of the duplex DNA they travelled down to reach the cruciform. (g) Cleavage of capped cruciforms extracts of E. coli

by

The experiments reported here strongly suggest that RecBCD enzyme cannot cleave pre-existing Holliday junctions in vivo. We therefore looked for another activity in E. coli that might perform that task. Fully capped cruciforms should be resistant to exonuclease degradation and hence should be good substrates for assaying cruciform cleavage activities in cell extracts. Extracts of E. coli were reacted with fully capped cruciforms with a 32P label at the EcoRI end. A strong band was seen,which appeared to result from nicks at B and C, together with weak

bands of sizes consistent with nicks at A and C or at B and D (Fig. 9). The main product, band was stable in the extract used in lanes 1 and 2, made from a recBC s&B mutant, but was degraded in the presence of RecBCD enzyme, exonuclease I (the sbcB gene product) or exonuclease VIII (expressed in sbcA mutants; data not shown). Appearance of the major band was dependent on the presence of the EcoK type I restriction enzyme in the strain. This band was abolished by mutation in either hsdS, the gene for the specificity subunit of the EcoK restriction enzyme (strains 8785 and V84. lanes 3 t,o lo), or by mutation in hsdR. t’he gene for the restriction subunit of the enzyme (strain V830. lanes 11 to 14). Both the hsdR and hsdS subunits are needed for the restriction nuclease activity (Yuan, 1981). The longer arms of the cruciform, derived from E. coli DNA, each contain a copy of t,he putative EcoK recognition sequence (Yuan, 1981), one 370 bases from the BamHI site and the other 230 bases from the EcoRI site (McKittrick, N. H.,

Action

qf RecBCD

Enzyme

Taylor, A. F., Bauer, C., Gardner, J. & Abcarian, P., unpublished results). The Ml3 DNAs used to make the cruciform were grown in an EcoK nonmodifying strain. Type I restriction enzymes recognize, and bind at, specific sequencesbut then cleave DNA at sites that may be several thousand bases away (Yuan, 1981). Cleavage can occur on either side of the recognition sequence (Yuan et al., 1980). In this experiment, one EcoK restriction enzyme may have bound to one of its recognition sequences, translocated DNA past itself and then cleaved the DNA when it met the barrier of the base of the cruciform. Alternatively, the cutting may have resulted from enzymes binding to each recognition site: translocating DNA past themselves and cutting when the two enzymes collide, a mechanism proposed by Studier & Bandyopadhyay (1988) for the cleavage of DNA by type I restriction enzymes. In the absence of EcoK restriction enzyme, however, weak double-strand cleavage activity by E. coli extracts was still seen. Two bands were produced, whose mobilities were consistent with diagonal cleavage of the cruciform at A and C or at B and D. This cleavage, of the type expected for an enzyme that resolves Holliday junctions, was seen in the absence of RecA protein, RecBCD enzyme or exonuclease I (Fig. 9, lanes 3 to 14). As the activity was present in a recBC sbcB strain (lanes 11 to 14), which typically acquire spontaneous sbcC mutations, it is most likely not the result of &CC gene product activity (Lloyd & Buckman. 1985). The identity of this cruciform-cleaving activity thus remains unclear.

4. Discussion The swapping of single strands of the same polarity between two homologous chromosomes can produce a Holliday junction (Fig. l), a postulated intermediate in many models of homologous recombination. Such a structure is flanked by homologous sequences, and hence the branch point in it can diffuse along the DNA (Thompson et al., 1976). Biochemical tests of postulated Holliday junction resolving enzymes have therefore used cruciform DNA structures as analogs of Holliday junctions. Initial experiments investigating the phage and yeast enzymes (see Introduction) used an inverted repeat in a circular duplex DNA: topological strain causes the inverted repeat to extrude into a cruciform structure. Such a molecule is devoid of DNA ends, which are required for RecBCD enzyme action. Non-diffusing cruciforms with DNA ends have been made by annealing four strands of DNA to form cruciforms that share virtually no homology between their arms (Mizuuchi et al., 1982: de Massy et al.. 1987). We show here that RecBCD enzyme can cut such a model Holliday junction in vitro. The most prominent reaction seen, the result of two diagonally opposed nicks in the structure as drawn in Figure 1, was one that would resolve genuine recombination intermediates into two “spliced” recombinant

on Cruciform

DNA

131

products or return them to their initial parental configuration. However, we also found that for both strands of the cruciform DNA to be cut, RecBCD enzyme molecules must approach the cruciform along more than one duplex DNA arm. As discussed below, this latter finding implies that RecBCD enzyme probably does not resolve pre-existing Holliday junctions in E. co&. The hypothesis (Thaler et al., 1988) that RecBCD enzyme nicks DNA at a constant rate, and hence would cleave a cruciform that impeded the travel of the enzyme. could explain resolution of the cruciform but would imply that the cruciform should be resolved equally in both directions. The phage T4 and T7 and the yeast enzymes apparently approach and recognize cruciforms from the “outside”: they can cut cruciforms in DNA molecules that are devoid of termini (Mizuuchi et al., 1982; de Massy et al., 1987; West & Kiirner, 1985; Symington & Kolodner, 1985). We detected, in extracts of E. co&, an activity capable of cutting fully capped cruciforms. The major activity in wildtype extracts cut off the labeled arm of the cruciform and was found to be the EcoK t’ype 1 restriction enzyme (Fig. 9). In the absence of EcoK, the extracts produced fragments consistent’ in size with the two diagonal cleavages of the cruciform. The activity was independent of RecA protein, RecBCD enzyme or exonuclease I. This act’ivity, which can resolve cruciforms devoid of termini, may be the same as a recA-independent activity previously discovered upon introduct’ion of figure 8 molecules (two circular plasmids fused by a Holliday junction) into E. coli. (West et al., 1983). This latter activity is, like the activity described here, independent of recB or sbcB (the gene for exonuclease 1) function (S. West, personal communication). The purification of the activity and the identification of the gene(s) encoding it may reveal a role for t,his activity in genetic recombination. RecBCD enzyme needed accessto the t’ermini of the cruciform for it to be able to resolve it. Purified RecBCD enzyme has several other activities on duplex DNA, all of which require that the enzyme have access to a flush or nearly flush duplex end. The double-stranded DNA exonuclease activity and the DNA unwinding activity of the enzyme both require DNA termini with flush or nearly flush ends (Prell & Wackernagel, 1980; Taylor & Smith, 1985). The nick produced by purified RecKCD enzyme on duplex DNA at Chi recombination hotspots also requires a duplex end on the DNA (Ponticelli et al.: 1985). Presumably a RecBCD enzyme molecule enters the end of the duplex DNA and travels along it, unwinding the DNA as it travels, encounters the cruciform or the Chi site and then nicks the DNA. Two phosphodiester bonds must, be broken to resolve a Holliday junction. The &ccharomyces enzyme makes the two nicks co-ordinately (Evans & Kolodner, 1988) but RecBCD enzyme, as shown here, resolves a cruciform by making the two nicks independently. When only a single end was available for the entry of RecBCD enzyme, only one

132

A. F. Taylor and G. R. Smith

strand of the cruciform was nicked (Fig. 8). When two or more ends were available for entry, the cruciform was resolved into two linear duplexes. The hairpin-shaped oligonucleotide caps that were used to prevent entry of RecBCD enzyme onto the duplex DNA ends would not appear to put any t,opological constraints on the DNA other than preventing the separation of the strands. It seems most likely then that the resolution of the cruciform is the result of separate enzyme molecules approaching the cruciform from separate directions. Both the principal nick observed here on encountering a cruciform and the nick made on meeting a Chi site are on t,he st.rand at, whose 3’ end the RecBCD enzyme had entered (Ponticelli et al.. 1985). Unwinding of duplex DNA by RecBCD enzyme also seems to result from the enzyme interacting principally with the strand at whose 3’ terminus it had entered the DNA (Braedt & Smith, 1989). However, results with different arms of the cruciform uncapped showed that the simple rule of “cut,ting the strand whose 3’ end was entered” cannot explain the specificity of double-stra,nd cutting. As the arms of cruciforms were only rarely cut off, an enzyme entering a given end presumably must travel to the base of the cruciform and then nick one! but not both, of the strands on which it is travelling. The DNA sequence around the base of the cruciform may dictate which strand of an arm is rut by an enzyme travelling along that, arm of the cruciform, and hence dictate the directionality of its resolution. The cruciform used in these experiments, a synthetic intermediate in A integrative recombination. contains 15 base-pairs of homology surrounded by non-homologous sequences. The Saccharomyces enzyme also shows directionality of cleavage on the substra.te used here: -9Oq/, of the cuts are on the BD axis, as defined in Figure 1 (Evans & Kolodner, 1988). Changing one to six of the bases in the homologous “core” of the sequence greatly changes t,he reaction products: in one case the cruciform is rendered uncleavable, while in other cases the directionality is abolished. The T7 enzyme shows modest directionalit,y of cutting with the substrate used in this paper, while the T4 enzyme shows symmetric cutting with a smaller 2 attachment site substrate containing the central part of the DNA used here (de Massy et al., 1987; Mizuuchi et al., 1982). The principal cut sites for the T7 and the Saccharomyces enzymes are all within three nucleotides of the region of homology in the cruciform (de Massy et al., 1987; Evans & Kolodner, 1988), and hence could represent cutting right at the base of the cruciform in one of its isomers (the base could be anywhere in the region of homology). It would seem fortuitous if the enzymes had preferred cut sites that happened to fall in the core region and which were altered by the core site mutants used. It seems more probable that, as demonstrated recently with small synthetic cruciforms (Duckett et al.. 1988), small changes in sequence can greatly affect the conformation of the cruciform, which can in turn bias the directionality

of cleavage. On this view the conformation might, also bias which strand on an arm of the cruciform was nicked by a RecBCD enzyme travelling along that arm. We have shown above that RecBCD enzyme has to approach a cruciform from more than one direction in order to cleave the cruciform. Genetic experiments with phage 1 and Chi sites suggest that RecBCD enzyme may not be afforded such an opportunity in E. coli. The chromosome of phage 2 replicates, m the recombination-deficient (red gam) phage mutants used, as covalently closed circles (Enquist & Skalka. 1973). Such circles are matured into packageable linear chromosomesby the phage‘s t,erminase protein, which recognizes and cuts at lambda’s cos (cohesive end site). The terminase protein binds to a site to the right of COB.at the left end of a linearized chromosome. as shown in Figure 10 and remains bound aft,er cleavage (Feiss & Widner, 1982). Hence the cleavage of cosprovides a unidirectional entry point for RecBCD enz.yme into the % chromosome (Stahl et al.. 19%). Lyt~itr crosses between two genetically marked ;1 phagel; may proceed via, a Holliday junction intermediate. Such an intermediate will necessarily have a cossite from each parent’ and hence, if both cos sites were cut, RecBCD enzyme molecules would have the opportunity to approach the cruciform along two of its arms and to subsequently cleave it. However, ReeBCD enzyme-dependent recombination also occurs between phage 2 and Adv. a L-derived autonomouslv replicating plasmid, which is devoid of 1’0ssites (itah et al.. 1982). In such a cross, as shown in Figure IO, RecBCD enzyme can enter from only one end of the solitary cos site and hence, as shown here, will not’ be able t,o cleave a pre-existing Holliday junction. The recombination observed is ReeBCD enzyme-dependent,, as shown by its stimulation by Chi. The frequency of RecBCD enzyme-dependent recombination between i and Ldv is comparable, per base-pair of homology, to that between II phages. It, would thus appear t,hat RecBCD enzyme alone cannot be responsible for the cleavage of pre-existing cruciforms in homologous recombination. A duplication of the cos site is necessary for the i chromosome t,o be packaged (Ross & Freifelder. 1976). Such a duplication is supplied, in these experiments, by the recombination-mediated dimerization of the 1 chromosome, before or after its recombination with the plasmid. As shown in Figure 10, this second recombination event does not provide a second avenue for RecBCD enzyme to approach the Holliday junction. A I*encoded gene product, rap, is essential for RecBCIJ enzyme-dependent recombination between R and some plasmids (Hollifield et al., 1987). The function of the rap product is unknown, but, the possibility that it might allow entry of RecBCD enzyme into circles cannot be excluded. As argued above, RecBCD enzyme cannot be solely responsible for the cleavage of prc-existing Holliday junctions in E. coZi. However, if RecBCD enzyme is involved in the formation of the Holliday

Action of RecBCD

aff

OF’

Enzyme on Cruciform

133

DNA

R I

0

Recombine with dv

Dimerize t lambda

P

Y Recombine with dv

Dimerize lambd I Cut co.5sites I Cut at cos site t

Figure 10. Postulated intermediates in the recombination of phage 1 and the plasmid Adv. A linear A molecule is shown at the top of the Figure, with relevant genes identified. Pairs of arrowheads denote the 2 halves of the cos site. and the stippled circle denotes the A terminase protein, which binds to, and remains bound to, the region shown when it cuts a cos site. Idv, always circular, is shown interacting with circular 1 DNA to form a figure 8 intermediat.e with the 2 DNAs joined by a Holliday junction. Only multimeric i genomes can be packed into mature phage. Dimerization of I either before or aft,er formation of the Holliday junction is shown. The subsequent rleavage of 1 or more cos sites allows access to the Holliday junction from only a single direction, since the terminase protein remains bound as shown.

junction, it may become entrapped within the junction or form the junction immediately behind itself (see Smith et al., 1984, Fig. 2) and ma.y under such circumstances be able to resolve it. We thank Kiyoshi Mizuuchi, Mary O’Dea, Martin Gellert, Claire Shurvinton, Frank Stahl and Steve West for allowing us to mention their unpublished results, and Niki McKittrick, Carl Bauer, Jeff Gardner and Peter Abcarian for the use of their nucleotide sequence of at@. We thank Bob Weisberg for kindly providing the Ml3 phage derivatives used, David Evans and Bernard de Massy for providing much needed encouragement and advice in the preparation of cruciforms and Stanley Brown, Claire Shurvinton, Karen Sprague and Bernard Weiss for bacterial strains. We thank our colleagues for their helpful criticisms of the manuscript. We thank the Hercules Powder Company, Wilmington, DE, for supplying nitrocellulose powder and Ernie Tolentino, of the Hutchinson Cancer Research Center, for keeping the DNA synthesizer working smoothly. This work was supported by NTH grant GM32194.

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