Cloning, Overexpression, Purification, and Characterization f the

THEJOURNALOF BIOLOGICAL

Vol. 269, No. 7, Issue of February 18, pp. 5187-5194, 1994 Printed in U.S.A.

CHEMISTRY

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Cloning, Overexpression, Purification, and Characterization of the Escherichia coli RuvC Holliday Junction Resolvase* (Received for publication, July 16, 1993, and in revised form, October 15, 1993)

Hazel J. Dunderdale, Gary J. SharplesS, RobertG. Lloyd*, andStephen C. West§ From the Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, U.K., and the Wepartment of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom

The RuvA and RuvB proteins have been purified (19-21) a n d The ruvC gene has been cloned into the plasmid pT7-7 shown to promote T7 410 promoter. Following the branch migrationof Holliday junctionsin under the control of the inductionwith isopropyl-1-thio-P-D-galactopyranoside,vitro (20, 22-26). The product of ruvC, the 19-kDa RuvC prothe 19-kDa RuvC protein was overexpressed 2030% to of tein, resolves Holliday junctions in vitro (27). Resolution was total cell protein.RuvC has been purified to homogene-demonstrated using: (i) synthetic Holliday junctions; (ii)recomity by a simple procedure involving precipitation from bination intermediates made by the RecA protein; and (iii) cruthecrudelysate,followed by threechromatographic ciform structures extruded from supercoiled plasmids (27,281. steps. The purified protein resolves synthetic Holliday Cleavage occurred by the introduction of two symmetrical nicks junctions (60 nucleotides in length) by cleavage at the close to the junction point(3, 27, 29). In the present work, we 3’-side of a phosphate group, to produce nicked duplexdescribe the cloning of the ruvC gene into a high level expresDNA. Under the same conditions no cleavage of linear sion plasmid and detail the purification of RuvC protein. We duplex or single-strandedDNA was detected. However, investigate the substrate specificity a n d cofactor requirements low levels of cleavage were observed with supercoiled and report the genDNA substrates, con- of the cleavage reaction catalyzed by RuvC form I and single-stranded circular eral biochemical and physical properties of the purified protein. sistent with the interaction ofRuvC withsecondary structures. Using synthetic Holliday junctions, we show EXPERIMENTALPROCEDURES that RuvC-mediated resolution requires Mg2‘ (10 mm) Enzymes and Reagents and exhibitsan alkaline pH optimum (pH9.0).No energy cofactors are needed. When RuvC was analyzed by gel Restriction enzymes wereobtained from New England Biolabs, Amfiltration and polyacrylamide gel electrophoresis, monopliTaq DNA po-lymerase from Perkin-Elmer-Cetus Instruments, calf meric and dimericforms of the protein were observed. intestinal phosphatase from Boehringer Mannheim, and T4 polynucleotide kinase from Pharmacia LKB Biotechnology Inc. T4 endonuclease VI1 (30) was a gift of Dr Bonies Kemper (University of Cologne). Terminal transferase, [U-~~PIATP, and [y-32PlATP wereobtained from AmHomologous recombination occursvia an intermediate struc- ersham Corp. ture, known as a Holliday junction, in which two DNA molBacterial Strains and Plasmids ecules are linked by a crossover (1). To complete the recombiE. coli strain BL21 (DE3) pLysS (31), plasmid pT7-7 (32), and plasnation event, resolutionof the Holliday junctionis required to restore the DNA to two discrete molecules.In Escherichia coli, mid pGS760 (5) are described elsewhere. The construction of plasmid an activity capable of performing Holliday junction resolution pGS775 is described under “Results.” in vitro was identified using cell-free extracts (21, and this Polymerase Chain Reaction activity was later found to beabsent i n ruvC m u t a n t s (3). A standard reaction contained oligonucleotidesA and B (100pmol of The ruvC gene is located withinthe ruv locus, at 41 min on each molecule), pGS760 (60 ng), and AmpliTaq DNA polymerase (5 the E. coli chromosome (4-6). The gene forms an operon with units). 30 cycles wereset upas follows: 40s denaturing at 92 “C, 1min orfZ6, which encodes a 26-kDa proteinof unknown function.A of annealing a t 45 “C, and 2.5 min of extension at 72 “C. The product second operon lying downstream of ruvC encodes two genes, was isolated by gel purification (Geneclean 11, Stratech). ruvA and ruvB (7, 8). The ruvA and ruvB genes are L e d DNA Substrates regulated and induced as p a r t of the SOS response to DNA in one of the three ruu damage (7-10). Cells carrying mutations The synthetic Holliday junction (4-Xl2) was prepared by annealing genes have similar phenotypes, withan increased sensitivity to four partially complementary oligonucleotides(each approximately 60 U V light, ionizing irradiation, and chemical mutagens(4, 11- nucleotides in length). DuplexDNA was prepared by annealing two Annealing was performedas described 14). In addition, in a recBCsbcA, recBCsbcBC, or recG genetic complementary oligonucleotides. background, ruu m u t a n t s are deficient in homologous recombi- (331, and the oligonucleotide sequences are listed in the accompanying nation (11, 15-18). In certain cases, the effect of ruu on the paper (34). The annealed substrates were 3ZP-endlabeled in the common strand (oligonucleotide2). Labeling was performedusing T4 polyphenotype of these multiple mutants is suppressed by recA nucleotide kinase and [y-32PlATP to give a 5’-end label or terminal mutations (151, suggesting that the ruuA, ruvB, a n d ruuC gene transferase and [ ~ ~ - ~ ~ P l d i dATP e o x yt o give a 3’-end label. Singleproducts are involved in a late step of recombination and the stranded and replicative form I (RFI)’ DNAof 4x174 were purchased from New England Biolabs. 3H-Labeled single-stranded 4x174 DNA recombinational repair of damaged DNA. was prepared as described (35). Unless stated otherwise amounts of DNA are expressed in mol of nucleotide residues. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked The abbreviations used are: RFI, replicative form I; DTT, dithio“aduertisement”in accordance with 18 U.S.C.Section1734solely to threitol; IPTG, isopropyl-1-thio-P-o-galactopyranoside; PAGE, polyindicate this fact. acrylamide gel electrophoresis;MES, 4-morpholineethanesulfonicacid; 5 To whom correspondence should be addressed. MOPS, 4-morpholinepropanesulfonicacid.

5187

5188

Characterization of RuvC Holliday Junction Resolvase Buffers

Lysis buffer contained 0.1 M Tris-HCl, pH 8.0, 2 m~ EDTA, and 5% (v/v) glycerol.R buffer contained 20 IILM Tris-HC1, pH 8.0, 1m~ EDTA, 1 m~ dithiothreitol ( D m ) and 10%(v/v) glycerol.TAE buffer contained 40 m~ Tris base, 1.1% (v/v)acetic acid, and 1 m~ EDTA. TBE buffer contained 89 m~ Tris base, 89 m~ boric acid, and 2 m~ EDTA. SDSsample loading buffer contained 0.125 M Tris-HC1,pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.01% (w/v) bromphenol blue, and 10% (v/v) p-mercaptoethanol. Cleavage buffer forRuvC and T4 endonuclease VI1 was 25 m~ Tris-HC1, pH 8.0, 10 m~ MgCl,, 100 pg/ml bovine serum albumin, and 1 m~ D m .

Time Course of Induction of RuvC Expression A 200-ml culture of BL21 (DE3) pLysSpGS775wasgrown with aeration at 37 "C in Luna broth containing 100 pg/ml carbenicillin and 50 p g / d chloramphenicol.At a cell density corresponding to A,, = 0.5, synthesis of RuvC protein was induced by the addition of isopropyl-lthio-p-D-galactopyranoside(IFTG) to 0.4 m ~ At . various times, 1.5-ml samples were removedand thecells pelleted by centrifugation. The cells were resuspended in 100 plof SDS-sample loading buffer and boiled for 3 min before analysis by SDS-PAGE.

chicken ovalbumin(44,000),horse myoglobin (17,000).and vitamin B,, (1,350). Protein was detected by measuring the absorbance a t 280 n m .

Gel Electrophoresis SDS-PAGE was carried out according to standard procedures using 13.5% gels (36). Unless stated otherwise, SDS-sample loading buffer was added to samples that were boiled for 4 min prior to loading. The standards (Bio-Rad) wererabbit muscle phosphorylase b (97,000),bovine serum albumin (66,200), hen egg white albumin (45,000), bovine carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and hen egg white lysozyme (14,400). Gels were stained with Coomassie Brilliant Blue or Rapid-Ag-Stain (ICN). Agarose gelelectrophoresis was performedusing TAE buffer and the DNA visualized by staining with ethidium bromide. For neutral PAGE, samples were electrophoresedthrough 6% polyacrylamide gels.For denaturing PAGE, the DNA products were heated to 95 "C for 3 min in TBE buffer containing 90% (v/v) formamide. Denatured samples were subjected to electrophoresis through 12% polyacrylamide gelscontaining 7 M urea. In both cases TBE buffer was used. The DNA was visualized by autoradiography. RESULTS

Construction of RuvC Overexpression Plasmid-In previous studies, we purified RuvC protein from plasmid pGS762 in Reactive Blue4-agarose and single-stranded DNA-cellulose were ob- which the ruvC gene was under control of the lacpromoter (27). tained from Sigma and phosphocellulose (P11) fromWhatman. A flow Induction of E. coli FB800 carrying pGS762 by IPTG led to low rate of 30 mVh was used with Reactive Blue4-agarose and phosphocellulose columns, and 20 mVh for the single-stranded DNA-cellulose col- level overexpression of RuvC protein ( 3 4 % of total cellular protein). To facilitate greater overexpression of RuvC, the ruvC umn.All column chromatography was carried out at 4 "C. gene was cloned into theplasmid pT7-7. A clone was generated Protein Concentrations by site-directed mutagenesis of ruvC, in which the upstream All protein concentrations were determined using a protein assay kit and downstreamsequences were altered. Two oligonucleotides (Bio-Rad) with bovine serum albumin as standard. Amounts of RuvC were constructed as primers for a polymerase chain reaction are expressed in moles of monomeric protein. (Fig. 1B). Oligonucleotide A corresponds to 34 bases of the 5' sequence of ruvC. It differs from the wild-type sequence at 6 Resolution Assay residues giving an EcoRI restriction site and an improved riReactions (20 pl) containing 5'-32P-labeledjunction DNA (150 n ~ ) , bosome binding site. Oligonucleotide B corresponds to 22 bases cleavage buffer,and RuvC protein were incubated for 30 min at 37 "C. of the 3' sequence of ruvC and differs from the wild-type se, the DNA was They werestopped by the addition of EDTA to 25 m ~ and quence at 2 residues giving an HindIII site. Using the two analyzed by neutral or denaturing PAGE followed by autoradiography. oligonucleotides a polymerase chain reaction product was generated from the plasmid pGS760 (ruvC+)(5). The product was Nonspecific Endonuclease Assays digested withEcoRI and HindIII and inserted into plasmid the Single-stranded or RFI 4x174 DNA (76 p)was incubated in cleav- pT7-7 (32). The resulting plasmid, pGS775, carries the ruvC age buffer (40 pl) a t 37 "C for 15 or 30 min, respectively,in thepresence gene under the control of the T7 410promoter (Fig. lA)and is of various amounts of RuvC. Reactions were stoppedby the addition of able to restore complete UV resistance to the four available EDTA to 25 m ~ and , the DNA was analyzed by electrophoresis through ruvC mutants.Confirmation that pGS775 carried thewild-type 1% agarose. ruvC gene was obtained by DNA sequencing.2 Plasmid pGS775 was transformed intoBL21 (DE31pLysS. A Analysis of Cleavage %-mini J'-ll?rmini-Reactions (60 pl) containing 5'-32P-end-labeled4-X12 time course of the induction of pGS775 following IPTG treatDNA (approximately 0.15 p)and RuvC (0.67 pd or T4 endonuclease ment is shown in Fig. 1C. Overexpression of the 19-kDa RuvC VI1 (300 units) were incubated in cleavage buffer for 30 mina t 37 "C. protein was apparent after 60 min of induction (Fig. lC, lane The DNA products were denatured by heating to 100 "C for 2 min and d ) . Maximal expression was achieved after 180-240 min, with the reactions split into two 30-pl aliquots. To each aliquot we added terminal transferase buffer (solution 1, Amersham 3'-end labeling kit). RuvC expressed to 20-30%of total cellular protein (Fig. lC, After a 20-min incubation at room temperature in the presence or lane g;Fig. 2 B , lane c; and data not shown). Purification of RuvC Protein-A summary of the purification absence of terminal transferase (10 units) and 2 m~ dATP (total volume , of 50 pl), reactions were stopped by the addition of EDTA to 20 m ~ and procedure is shown in Fig. 2 A . Two 1-liter cultures of E. coli the 32P-labeledproducts were analyzed by denaturing PAGE. strain BL21 (DE3) pLysS carrying pGS775 (ruuC') were g r o w , 5'-'ll?rmini-3'-32P-End-labeled 4-X12 DNA(0.15 p)and RuvC (0.17 with aeration, at 37 "C in Luna broth containing 100 pdml p ~ were ) incubated in cleavage buffer (80pl) for 30 min at 37 "C. The reaction was stopped and the DNA denatured by heating to 100 "C for carbenicillin and 50 pg/ml chloramphenicol. At a cell density pl) were incubated in the presence or absence of calf corresponding to Asso = 0.5, IPTG was added to 0.4 m~ and 2 min. Aliquots (20 intestinal phosphatase (0.1 unit). After 40 min at 37 "C,EDTA was incubation continued for 3.5 h. After chilling on ice, induced added to 50 m ~ Following . ethanol precipitation, DNA was kinased cells (Fig. 2 B , lane c)were collected by low speed centrifugation using T4 polynucleotidekinase (36)in a reaction containing 100 p~ ATP. and resuspended in20 ml of lysis buffer, giving a total volume The 32P-labeledproducts were analyzed by denaturing PAGE. of 30 ml. The cells were fast-frozen on dry icdethanol and stored at -20 "C. Gel Filtration When required, thecells were thawed, and0.2 volume of 5 M RuvC (30 pg) was dialyzed for 3 h against R buffer supplemented NaCl, 0.01volume of 100 IMID'IT, and 0.01 volume of 10% (v/v) with 10 m~ MgClz and 150 m~ NaCl and applied to a Superose 12 HR Triton X-100 were added. The high salt concentration was necChromatography

10/30 fast protein liquid chromatography column (Pharmacia). The column was precalibrated using molecular mass standards (Bio-Rad) consisting of bovine thyroglobulin (670,000), bovine y-globulin (158,000).

N. F. P. Hagan and G. J. Sharples, data not shown

5189

Characterization of RuvC Holliday Junction Resolvase A

HiDdm

C

M 0 30 60 90 120 180min ~~

97 kD-

66 kD-.

bfa

45 kD-

31 kD" " "

B

RBS

EcoRl 5 ' -GGCCIGCTA"GCGTGATG-3

'

S'-GGCCTGCTAAI\CAGCAAAA$-G:CGCGTGATG-3'

... . . . ." ""3 "

- "b

a

3'-bGCCGACT&G&AAGGTGAAGd-S0

3'-GGCCGACTTCGWGGTGAGG-S'

c

d

e

.

f

g

wild type oligo B

Hindln

FIG.1. Overexpression of RuvC protein.Panel A shows the plasmid pGS775, in which the ruuC gene wasplaced under thecontrol of the T7 ~$10promoter inpT7-7. Panel B , the ruuC clone was generatedby polymerase chain reaction using two synthetic oligonucleotides. Oligonucleotide A (5'of ruuC) and oligonucleotide B (3' of m u C ) differ from the wild-type sequence a t several bases (indicated by asterisks). These alterations produced EcoRI and Hind111 sites anda modified ribosome binding site.Panel C shows the time courseof the induction of pGS775 (ruuC+).IFTG was addedat 0 min to a cultureof BL21 (DE3)pLysS pGS775. At the times indicated, samples were taken and analyzed by SDS-PAGE (lanes b-g), as described under "ExperimentalProcedures." Molecular mass markers (lane a ) and theposition of the RuvC protein are indicated. Thegel was stained with Coomassie BrilliantBlue.

Reactive Blue 4-agarose (Fraction 11) (Elute 0.6.07 M KCI)

31 kD.

1 Phosphocellulose (Fraction 111) 1 IV) ssDNAcellulose

21 kD.

(Elurn 0 . 5 . 0.7 M KCI)

(Fraction

(Elurn 0.15.0.3 M KCI)

31 kD21 kD-

.RuvC

RuvC

I4 kD-

I4 kD

a

b

c

d

e

I

g

FIG.2. Purification of RuvC protein. Panel A , summary of purification. Panel B , SDS-PAGE showing the purification of RuvC. Lune a, molecular mass markers; lane 6 , cellular proteins immediately before IFTG induction; lane c, cellular proteins 3.5 h after induction; lane d, resuspension of proteins precipitated from crude lysate (fraction I); lanes e-g, fractions eluted from Reactive Blue 4-agarose (fraction 11). phosphocellulose (fraction 111), and single-strandedDNA-cellulose (fraction IV),respectively. Lane g contains 6 pg of protein. The gel was stained with Coomassie Brilliant Blue. Panel C, silver-stained SDS-polyacrylamide gel. Lane h, molecular mass markers; lanei, purified RuvC (0.2 pg).

essary to prevent precipitation of RuvC. Cells were lysed by three rounds of freezing and thawing, and the lysate was centrifuged for 60 min at 38,000 rpm ina Beckman Ti-45 rotor. The clear supernatant (36 ml) was dialyzed for 3 h against R buffer supplemented with 0.1 M KCI. During dialysis a heavy precipitate formed. The precipitate wascollected by centrifugation at 15,000 rpm for 10 min, washed with R buffer containing 0.1 M KCI, and recentrifuged. The pellet was resuspended by addition of 90 ml of R buffer containing 0.5M KCl. SDS-PAGEof the resuspended precipitate (fraction I) showed it was more than 90% pure (Fig. 2 B , lane d ) . This fraction is sufficiently pure for physical analysis of RuvC. To remove minor contaminants and obtain protein for biochemical analysis, RuvC was purified further by three chromatographic steps. Fraction I (90 ml; approximately 0.6 mg of

proteidml) was applied to a Reactive Blue 4-agarose column (1.6 x 14.0 cm, 28-ml bed volume) equilibrated with R buffer containing 0.5 M KCI. The column was eluted with a 400-ml gradient of 0.5-1.75 M KC1 in R b ~ f f e rFractions .~ eluted from the column were assayed in two ways; (i) the peak of RuvC protein was identified bySDS-PAGE or (ii) the peak of resolvase activity was determined using 32P-labeled synthetic Holliday junctions. Resolution resulted in the formation of nicked duplex DNA, as detected by neutral or denaturing For the purification described here the lyophilized powder form of Reactive Blue 4-agarose (Sigma,R8754) was used. In subsequent purifications, the matrix was purchaseda s a suspension (Sigma,R2507) to which RuvC binds with a lower affinity. This makes it necessary to dialyze fraction I against R buffer containing 0.3 m~ KC1 before loading and use a gradientof 0.3-1.25 M KC1 in R buffer.

Characterization of RuvC Holliday Junction Resolvase

5 190

A

97kD-

-

d

M FII FT 10 11 12 13 14 15 16 17 18 19

z

A

a L

66kD-

RuvC (1

45 kD31 kD-

RuvC

a

b

c

d

e

f

g

h

i

j

k

l

m

a

h

c

d

e

l

g

B

B

substrate

-" n o p q r s t u v w x y

RuvC (pM) 0 0.1 0.2 0.5 0.8 1.6 3.2 M

SSDNA-

-cleavage product

FIG.4. Endonuclease assay.Panel A, RFI +X174 DNA (76 phi) was incubated with the indicated concentrations of RuvC (lanes a-e) as described under "ExperimentalProcedures." The markers are PstI-linearized RFI 6x174 DNA (lane f ) and a 1-kilobase DNA ladder (lane g). Panel B,single-stranded (ss) 6x174 DNA (76 phi) was incubated with RuvC a s indicated (lanes h-n). Lane 0, RFI/II 6x174 DNA marker. Reaction products were analyzed by electrophoresis through 1%agarose and visualized by ethidium bromide staining.

FIG.3. Coelution of RuvC protein with Holliday junction resolution activity. Panel A, elution profile of RuvC from a phosphocellulose column a s determined by SDS-PAGE. Lane a , molecular mass markers; lane b, sample applied to column (fraction 11); lane c, column flow-through ( F T ) ;lanes d-rn, fractions eluted from the column (not all fractions are shown). The gel was stained with Coomassie Brilliant Blue. Panel B, the same fractions were assayed for resolution of a 5'-32P-end-labeled syntheticHolliday junction (4-X12), as described un- single-stranded DNA-cellulose column (1.0 x 2.5 cm, 2-ml bed der "Experimental Procedures." Labeled reaction products were anavolume) and elutedwith a 40-ml gradient of 0.05-0.5 M KC1 in lyzed by denaturing PAGE and visualized by autoradiography. Lane n, R buffer. Again the RuvC protein and resolution activity were fraction 11; lane 0 , column flow-through; lanes p-y, fractions eluting found to coelute. The peak fractions (0.15-0.3 M KCI) were from phosphocellulose.

pooled and glycerol added to 50% (fraction IV Fig. B, lane g). PAGE followed by autoradiography (29). Fraction IV was homogeneous as determined by SDS-PAGE The peak of RuvC protein eluting from Reactive Blue 4-aga- followed by silver staining (Fig. 2C, lane i ) .After fast freezing rose (0.6-0.7 M KCI) was pooled and dialyzed against R buffer on dry ice/ethanol the protein was stored in aliquotsa t -70 "C. containing 0.2 M KC1 for 3 h (fraction 11). At this stage, RuvC Working stocks were stored a t -20 "C. The totalyield of RuvC appeared as a single band by SDS-PAGE (Fig. 2, lane e ) . How- protein was 6.3 mg in 10 ml. The pure RuvC appears to be ever, the presence of contaminating nuclease activity was re- unusually stable. After incubation at 37 "C for 14 h, we were vealed by a loss of end label during the resolution assay (data unable to detect degradation of the protein by SDS-PAGE, and not shown). Fraction I1 (50 ml; approximately 0.5 mg of proteid there was little or no loss of activity when tested in the resoml) was then applied to a phosphocellulose column (1.0 x 12.7 lution assay (data not shown). cm, 10-ml bed volume) and eluted with a 200-ml gradient of Substrate Specificity of Purified RuvC-To determine 0.2-1.0 M KC1 in R buffer. SDS-PAGE revealed a peak of protein whether RuvC possesses any nonspecific nuclease activity, in fractions 14-17 (Fig. 3A, lanes h-k) which coeluted with the various amounts of the protein were incubated with superpeak of resolvase activity (Fig. 3B, lanes t-w). Although the coiled RFI 4x174 duplex DNA. Reaction products were anaprotein appeared homogeneous a t this stage, overexposure of lyzed by agarose gel electrophoresis followed by ethidium brothe autoradiogramshown in Fig. 3B revealed trace amountsof mide staining. At RuvC concentrations of up to 1.4 p~ (i.e. 1pg a nonspecific nuclease activity in fractions 16-19, overlapping of RuvC/pg of RFI DNA) we were unable to detect cleavage of the RuvC peak. Rather thanaccept a considerable loss of RuvC the substrate(Fig. 4 A , lunes b d ) . However, at a RuvC concenby discarding the contaminated fractions, a third chromato- tration of 4.2 PM, which conesponds to 1 RuvC monomer/l8 graphic step was used to remove the nonspecific nuclease. nucleotides, we observed the appearance of trace amounts of The peak fractions from phosphocellulose (eluting between linear DNA product (Fig. 4A, lune e ) . 0.5-0.7 M KCI) were pooled and glycerol added to50% to allow To determinewhether RuvC protein possesses singleovernight storage at -20 "C without freezing. The following day stranded endonuclease activity, varying amounts of RuvC were the glycerol was removed by dialysis for 3 h against R buffer incubated with single-stranded circular 4x174 DNA. This DNA containing 50 mM KCI, giving fraction I11 (Fig. 2B, lane fl. substrate provides a sensitive assay for endonuclease activity Fraction I11 (20 ml; approximately 0.4 mglml) was applied to a since the introduction of two nicks/DNA molecule (5,382

A


I

5191

not form any secondary structure. Reactionscontained an equal numberof substrate molecules, and increasing amounts ofRuvC. The DNA products were analyzed by both neutral (Fig. 5 A ) and denaturing(Fig. 5B)PAGE. By neutral PAGE, we observed that RuvC cleaved the junction to give products which migrated in theduplex position (Fig. 5A, lanes c and d ) . Dena-junction turing PAGE of the same samples revealed that this occurred by specific nicking of the DNA resulting in a single labeled cleavage fragment (Fig. 5B, lanes o and p ) . Under identical conditions and RuvC concentrations, we were unable to detect cleavage or degradation of the duplex or single-stranded DNA by neutral or denaturing PAGE (Fig. 5, lanes e 4 and q-x). Fig. 5 indicates that RuvC has a high specificity for the junction DNA, with little or no activity on double- or singlestranded DNA. The apparentincongruity of these results with those presented in Fig. 4 can be explained in terms of secondary a b c d e f g h i j k l structure. Previously, Iwasaki et a1 (28) showed that RuvC cleaves the cruciform structure inpUC4 by the introduction of nicks at the base of the cruciform to produce linear duplex DNA. Since RFI 4x174 DNA contains a palindromic sequence B junction duplex (single-strand that can extrude intoa cruciform (37), the small amount of RFI RuvC (nM) 0 5 25125 0 5 25 125 0 5 25 125 4x174 DNA cleaved may be a result of interaction of RuvC with - subsaate thisstructure. Cleavage of single-strandedcircular 6x174 DNA could also be structure-specific sinceinteractions between regions of complementary DNA sequence could lead to the for- product cleavage mation of junction-like structures. Buffer Requirements of RuuC--To determine the buffer requirements of RuvC, cleavage of the synthetic Holliday junction was assayed under various reactionconditions. The results are shown in Fig. 6.RuvC activity was stimulated by alkaline pH, with optimum resolutionoccurring at pH 9.0 (Fig. 6A).The amount of cleavage obtained at more physiological pH (7.5) was about 17% of that observed at the optimum. RuvC showed a m n o p q r s t u v w x requirement for divalent cations (Mg2+) with an optimum at FIG.5. Cleavage specificity of RuvC. Reactions (40 pl)containing 5-10 mM (Fig. 6B). M$+ could be replaced by Mn2+but not by 38 n~ synthetic Holliday junction (4-Xl2)DNA (lanes a d and m-p), 19 m linear duplex DNA (lanes e 4 and q-t), or 9.5 m single-stranded Cu2+,Zn2+,Co2+,or Ca2+.4Resolvase activity was inhibited by salt, with 50% inhibition observed at 85 mM KC1 (Fig. 6C). We linear DNA (lanes i-l and u-x) were incubated with the indicated amounts of RuvC. 5'-32P-End-labeledoligonucleotide 2 was common to failed to observe any stimulationof activity by inclusion ofATP, all three substrates. After 60 min at 37 "C, the reactions were stopped spermidine or potassium glutamate (datanot shown). with EDTA and the labeled products visualized by autoradiography Characterization of the Nick Introduced by RuuC--To deterfollowing neutral (panel A ) or denaturing (panel B ) PAGE. mine the nature of the 3"terminal group created by RuvC a t nucleotides) results inloss of the substrate band as detected by the site of resolution, the cleavage products of a 5'-32P-end agarose gel electrophoresis followed byethidium bromide stain- labeled junction were heat denatured and treatedwith termiing. At RuvC concentrations below 0.5 PM, we observed no de- nal transferase. Terminal transferase catalyzes the addition of tectable cleavage of the substrate DNA (Fig. 4 B , lanes i d ) . deoxynucleotides to the 3'-end ofDNA substrates only if a However, at concentrations above 0.8 p (? 1 RuvC hydroxyl group is present. Upon incubation of the cleavage monomer/95 nucleotides), we observed loss of the substrate, products (Fig. 7A, lane c ) with terminal transferase,a ladderof characteristic of single-stranded DNA endonuclease activity DNA fragments was produced (lane d ) , demonstrating the (Fig. 4 B , lanes 1-n). Since the gel assay is incapable of deter- presence of a 3"hydroxyl group at the siteof RuvC resolution. mining the extentof DNA digestion, reactions containing uni- As a control, similar reactions were carried outusing cleavage formly 3H-labeled single-stranded 4x174 DNA were analyzed fragments produced by T4 endonuclease VII. This enzyme for the releaseof acid-soluble counts following incubation with cleaved 4-X12 DNA at a number of sites throughout the hoRuvC. At concentrations of RuvC which promoted the complete mologous core to give three major and several minor products loss of the single-stranded circular DNA, as measured by gel (Fig. 7A, lane e ) . Treatment with terminal transferase again electrophoresis (Fig. 4B,lanes m-n), we observed that less than resulted in a ladder of DNA fragments (Fig. 7A, lane f ). To determine the nature of the group at the 5'-terminus of 0.1% of the total 3H-labeled DNA was made acid-soluble (data not shown). These experiments indicate that RuvC does not the nick, the junctionwas labeled at the3'-end of oligonucleopossess any exonuclease activity and interactswith the single- tide 2. After incubation with RuvC, the DNA products were stranded circularDNA to introduce a small numberof endonu- heat denatured and treated with calf intestinal phosphatase, cleolytic incisions. which removes 5"phosphate groups from DNA. Phosphatase To investigate thespecificity of the endonuclease activity of treatment of a DNAmolecule possessing a 5"phosphate should RuvC protein, we compared its activity on a synthetic Holliday result in a shift of mobility, as detected by denaturing PAGE, junction (4-X12), linear duplex DNA and a single-stranded oli- because of the removal of a charged group. As seen in Fig. 7B, gonucleotide (Fig. 5). All substrates were of the same length treatment of the resolution products with calf intestinal phosand contained a common DNA strand (5'-32P-end labeled oli- phatase resulted in a slight reduction in the mobility of the gonucleotide 2). Oligonucleotide 2 was designed such that it possessed no internal complementary sequences and so could R. Shah, R. J. Bennett, and S. C.West, unpublished observations. RuvC (nM)

junction duplex Jsinglc-strand 0 5 25 125 0 5 25 12 0 25 5125 -origin

1

I

I

5192

Characterization of RuvC Holliday Junction Resolvase 1.2 1 .o

p3 0.8 1

-

1..I_

I

0 0.2 .0

4

5

6

7

I 8

9

1011

PH 1.2 r 1.0

I

cn

8“

3 0.8

-p 0 0

.z

0.5

0.2

0.0

0

10 :!O 30 40 50 60 MgCI, concentration (mM)

applied to a gel filtration fast protein liquid chromatography l l ~ and 150 column in R buffer supplemented with 10 ~ MgC12 mM NaCl. Measurement of the absorbance at 280 nm resulted in the elutionprofile seen inFig. 8 A . By comparison with protein standards analyzed in parallel (Fig. 8B), it was determined that the protein peak corresponded to a molecular mass of around 14 kDa. Upon SDS-PAGE of fractions collected during gel filtration, we observed that the elution of RuvC protein coincided exactly with the peak seenin the absorbanceprofile (data notshown). Sincethe predicted mass of RuvC is 18.7 kDa (5,61, the gel filtration data indicate thatRuvC is monomeric under these conditions. SDS-PAGE analysis of RuvC led to the observation of multiple bands on a gel, as seen in Fig. 9. The three major bands have been termed I,11, and 111. When denatured by dilution in 2% SDS and 10 mM D m , RuvC ran as a single band (11)of about 20 kDa (Fig. 9, lane a ) . However, after dilution in R buffer lacking DTT, we observed two additional major bands (Fig. 9, lane b ) . Band I corresponds to a molecular mass of around 39 kDa, consistent with the predicted mass of a RuvC dimer. Band I11 ran slightly ahead of the RuvC monomer, at around 17 kDa, presumably because of incomplete unfolding. We also observed 56 kDa and a doublet at 36 kDa). several minor bands (70 and When RuvC was diluted in the same buffer containing 1 l ~ l ~ DTT, we observed the unfolded and partiallyunfolded forms of monomeric RuvC, with only a trace of the 39-kDa dimer (band I) (Fig. 9, lane c ) . From these results we conclude that in the of the RuvC protein presence of a reducing agent, the majority is monomeric. However, in its absence, RuvC protein is found in both monomeric and dimeric states.

DISCUSSION In this paper we describe the construction of a plasmid in which the ruvC gene wasplaced under the control of the powerful T7 410 promoter. Following induction, RuvC protein was expressed to20-30% of total cell protein. By lysing thecells in the presence of 1M salt we were ableto keep most of the RuvC in solution and allow its isolation from the crude lysate. The first step in the purification procedure, precipitation of RuvC by dialysis of the crude lysate to low salt, yields protein thatis 0.0 > 90% pure. At this stage, after a very simple andquick proce0 100 200 300 dure, the protein sufficiently is pure for physical analysis. FurKC1 concentration (mM) ther purification through three chromatographic stepsyielded FIG.6. Effect of buffer conditions on RuvC-mediated resolu- approximately 6 mg of > 99% pure RuvC from 2 liters of culture. tion activity. Panel A, resolution activity was assayed as described under “Experimental Procedures,” over a pH range, by replacing the The methoddescribed here provides a significant improvement Tris-HC1, pH8.0, in the cleavage buffer with the following buffers(at 40 over our previous purification scheme, which utilized a comm): sodium acetate, pH 5.0 and 5.5; MES, pH 6.0 and 6.5; MOPS, pH paratively poor expression system (27), and thatof Iwasaki et 7.0 and 7.5;Tris-HC1,pH 8.0, 8.5, and 9.0; glycine, pH 9.5 and 10.0. al. (28), in which over 70% of the RuvC was notrecovered from Panel E , effect of MgCl, concentration. Panel C,effect of salt. In panels B and C the buffer contained Tris-HC1, pH8.0, at 40 m. All reactions the cells. Using small synthetic DNA molecules, made by annealing contained RuvC (34 m)and 5’-3ZP-end-labeled Holliday junction 4-X12 (0.15 w).The products of resolution were analyzed by autoradiography oligonucleotides, we investigated the substrate specificity of following denaturing PAGE and quantitated using a Molecular Dynam- RuvC. We found that the purified protein cleaves synthetic ics model 300 laser densitometer and ImageQuant software. Cleavage is Holliday junctions to form nicked duplex products. At similar defined relative to the maximum observed cleavage. RuvC concentrations, we were unable to detect cleavage of duplex or single-stranded DNA substrates. The resolution accleavage fragment (compare lanes h and j ) .To confirm that this tivity was optimalat alkaline pH and required divalent cations mobility shift was causedby the loss of a phosphate group, the (5-10 mM Mg2+). RuvC resolved the synthetic junctions by DNA was treated further with T4polynucleotide kinase to re- cleavage at the 3’-side of a phosphate, leaving 5“phosphate store the terminal phosphate. The migration of the DNA frag- and 3’-hydroxyl groups at the incision termini. The resulting nicks may therefore act as substrates for DNA ligase (29). ment returned to the originalposition (Fig. 7B, lane K). In addition to cleavage the of Holliday junctions, we observed These results indicate that RuvC cleaves DNA at the 3’-side that RuvC cleaved RFI 4x174 DNA with low efficiency to proof the phosphate group to generate a nick with 5”phosphate and 3’-hydroxyl termini. Elsewhere, we show that the nicked duce linear duplex DNA. Since 4x174 DNA contains short palduplex resolution products formed by RuvC can be ligated ef- indromic sequences capable of cruciform extrusion (37), it is likely that linearization results from resolution of a cruciform ficiently by E. coli DNA ligase (29). Physical Analysis of RuuC-To determine the native molecu- structure asdescribed previously (28). Similarly, the fragmenlar weight of RuvC, approximately 30 of pgpurified protein was tation of single-stranded circular 4x174DNA byRuvC is likely

-

Characterization of RuvC Holliday Junction Resolvase A

- - - +

RuvC T4endo VI1 terminal transferase

+ + - - - + + - +I - +

.

RuvC - + + + +

B

calf intestinal phosphatase T4 polynucleotide kinase substrate'

1

RuvC cleavage product

I

endo VI1 cleavage products

5193

dephosphorylated productL cleavage product'

-

- - + + - + - +

"

g h i j k a b c d e f FIG.7. Analysis of cleavage termini. Panel A, 3"termini. 5'-32P-End-labeled synthetic Holliday junction DNA (4-Xl2) was incubated alone (lanes a and b ) ,with RuvC (lanes c and d ) , or with T4 endonuclease VI1 (lanes eand f )a s described under "ExperimentalProcedures." The products were heat denatured, and aliquots were treated with terminal transferase (lanes 6, d, and f )or left untreated (lanes a , c, and e ) as indicated. The VI1 are indicated. Panel B , 5'4ermini. 3'-"P-End-labeled junction DNA(4-Xl2) was major cleavage products formed by RuvC and T4 endonuclease incubated alone (lane g ) or with RuvC (lanes h-k) a s described under "ExperimentalProcedures." The products wereheat denatured and aliquots incubated in the presence (lanesj and k ) or absence(lanesg-i) of calf intestinal phosphatase.DNA was precipitated and some samples (lanes i and k ) treated with T4 polynucleotide kinase in the presence ofATP. Reaction products were analyzed by denaturing PAGE and visualized by autoradiography.

A

8

670 kD I58 kD 44 kD I7 kD

4

4

4

b

97kD-

I .35 kD

66 kD45kD31kD-

4

6

-

M -

a

b

C

. I

" I

Absorbance 280 nm

2 0 15 25 20

30 35 40 Fraction Number

50

45

J

.35 m

2.42.22.01.81.61.41.2 VeNO FIG.8. Gel filtration. RuvC was applied to a Superose 12 fast protein liquid chromatography column in R buffer containing m10 M MgClz and 150 m~ NaCI, a s describedunder"ExperimentalProcedures." Panel A, elution profile; panel B , determination of the relativemolecular mass of RuvC by comparison with molecular mass standards. Absorbance a t 280 nm is expressed in arbitrary units.

to be caused by cleavage of junction-like structures formed by the interaction of complementary sequences. In the accompanying paper, we describe the substrate specificity of RuvC protein in further detail(34).

FIG.9. PAGE of RuvC protein. Samples of RuvC (5 pg) were applied to an SDS-polyacrylamide gel without boiling under a variety of buffer conditions: lane a, in SDS-sample loading buffer except that P-mercaptoethanol was replaced by 10 mM D m ,lane b, in R buffer wthout D m ,lane c, in R buffer (1 m~ Dll'). The positions of the molecular mass standards are indicated. Bands I, 11, and 111 are explained in the text (see "Results"). The gel was stained withCoomassie Brilliant Blue.

The subunit structure of RuvC protein has been investigated bygel filtration and PAGE. Upon gel filtration, the 19-kDa RuvC protein eluted as a single peak corresponding to a molecular mass of about 14 kDa. These results contrastwith those presented by Iwasaki et al. (28), who reported that RuvC behaves as a dimer upon gel filtration. However, the former studies were performed a t higher protein concentrations than those used in the experiment of Fig. 8. When we applied RuvC protein to anSDS gel without prior additionof a reducing agent (or boiling), we observed the presence of a band (39 kDa) that corresponded to theexpected mass of a RuvC dimer. This band was not observed when a reducing agent was added. From these datawe suggest that the nativeform of RuvC is a dimer that can be easily dissociated into the monomeric form. Consistent with this proposal, analyses of RuvC-Holliday junction complexes by band shift assaysindicate that thebound form of RuvC protein is d i m e r i ~ . ~ REFERENCES 1. Holliday. R. (1964) Genet. Res. Camb. 5,282404 2. Connolly, B.,and West, S. C. (1990)Proc. N d . Acad. Sei. U.S. A. 87,84768480

R. J. Bennett and S. C. West, unpublished data.

5 194


3. Connolly, B., Parsons, C. A,, Benson, F. E., Dunderdale, H. J., Sharples, G. J., Lloyd, R. G., and West, S . C. (1991) Proc. Natl. Acad.Sci. U.S.A. 88, 60634067 4. Sharples, G . J., Benson, F. E., Illing, G. T., and Lloyd, R. G . (1990)Mol. & Gen. Genet. 221,219-226 5. Sharples, G . J., and Lloyd, R. G . (1991) J. Bacteriol. 173, 7711-7715 6. Takahagi, M., Iwasaki, H., Nakata, A,, and Shinagawa, H. (1991)J. Bacteriol. 173,5747-5753 7. Benson, F. E.,Illing, G. T., Sharples, G . J., and Lloyd, R. G . (1988) Nucleic Acids Res. 16, 1541-1550 8. Shinagawa, H., Makino, K., Amemura, M., Kimura, S . , Iwasaki, H., and Nakata, A. (1988) J. Bacteriol. 170, 4322-4329 9. Shuninton, C. E., and Lloyd, R. G . (1982) Mol. & Gen. Genet. 186,352355 10. Walker, G . C. (1985)Annu.Reu. Biochem. 64,425-458 11. Lloyd, R. G., Benson, F. E., and Shurvinton, C. E. (1984) Mol. & Gen. Genet. 194,303309 12. Otsuji, N., Iyehara, H., and Hideshima, Y.(1974) J . Bacteriol. 117, 337344 13. Sargentini, N. J., and Smith, K. C. (1989) Mutat. Res. 215, 115-129 14. Stacey, K. A,, and Lloyd, R. G . (1976) Mol. & Gen. Genet. 143,223-232 15. Benson, F., Collier,S., and Lloyd, R. G . (1991)Mol. & Gen. Genet. 225,266-272 16. Lloyd, R. G . (1991) J. Bacteriol. 173, 5414-5418 17. Lloyd, R. G., Buckman, C., and Benson, F. E. (1987) J. Gen. Microbiol. 133, 2531-2538 18. Luisi-DeLuca,C., Lovett, S . T., and Kolodner, R. D. (1989) Genetics 122,269278 19. Iwasaki, H., Shiba, T., Nakata, A., and Shinagawa, H. (1989) Mol. & Gen. &net. 219,32a331 20. Shiba, T.,Iwasaki, H., Nakata,A., andShinagawa, H. (1991)Proc. Natl. Acad. Sci. U.S. A. 88,844543449

21. Tsaneva, I. R., Illing, G. T., Lloyd, R. G., and West, S . C. (1992) Mol. & Gen. Genet. 235, 1-10 22. Tsaneva, I. R., Muller, B., and West, S . C. (1992) Cell 69, 1171-1180 23. Parsons, C. A,, Tsaneva, I., Lloyd, R. G., and West, S . C. (1992) Pm. Natl. Acad. Sci. U.S. A. 89,5452-5456 24. Parsons, C. A,, and West, S . C. (1993) J. Mol. B i d . 232, 397405 25. Muller, B., Tsaneva, I. R., and West, S . C. (1993) J. B i d . Chem. 266, 1717% 17184 26. Muller, B., Tsaneva, I. R., and West, S . C. (1993) J. Biol. Chem. 266, 1718517189 27. Dunderdale, H. J., Benson, F. E., Parsons, C. A,, Sharples, G. J., Lloyd, R. G., and West, S . C. (1991)Nature 364,50&510 28. Iwasaki, H., Takahagi, M., Shiba, T., Nakata, A,, and Shinagawa, H. (1991) EMBO J. 10,43814389 29. Bennett, R. J.,Dunderdale, H. J., and West, S . C. (1993) Cell 74, 1021-1031 30. Kosak, H. G., and Kemper, B. W. (1990) Eur: J. Biochem. 194, 779-784 31. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and DubendorfF, J. W. (1990) Methods Enzymol. 185,6&88 32.Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad.Sci. U.S. A. 82, 1074-1078 33. Parsons, C. A,, Kemper, B., and West, S . C. (1990) J. B i d . Chem. 265, 92859289 34. Benson, F. E., and West, S . C. (1994)J. Biol. Chem. 269,5195-5201 35. West, S . C., Cassuto, E., Mursalim, J., and Howard-Flanders, P. (1980) Proc. Natl. Acad. Sci. U.S. A. 77, 2569-2573 36. Sambrook, E. F., Fritsch, E. F., and Maniatis, T.(1989) Molecular Cloning: A Luboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 37. Lilley, D. M. J. (1981) Nucleic Acids Res. 9, 1271-1289