Proc. Natl. Acad. Sci. USAVol. 86, pp. 2627-2631, April 1989Biochemistry

Identification and purification of a single-stranded-DNA-specificexonuclease encoded by the recJ gene of Escherichia coli

(genetic recombination/DNA repair/RecF pathway)

SUSAN T. LOVETT AND RICHARD D. KOLODNER*Department of Cell and Molecular Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; and Department of Biological Chemistry andMolecular Pharmacology, Harvard Medical School, Boston, MA 02115

Communicated by Charles C. Richardson, January 9, 1989

ABSTRACT The Escherichia coli recJ gene product wasoverproduced using a plasmid that carries the recJ genedownstream of a strong regulatable promoter and a strongribosome-binding site. Overexpression of recJ produced aconcomitant increase in the levels of single-stranded-DNA-specific nuclease activity present in crude cell extracts. Thisnuclease activity was purified to homogeneity and found toreside in a 60-kDa polypeptide. This polypeptide was inducedwith recJ overexpression and had the size and N-terminalamino acid sequence identical to the predicted RecJ proteinsequence. The RecJ nuclease degraded linear single-strandedDNA but did not have exonuclease activity on linear double-stranded substrates or endonuclease activity on either single-stranded or double-stranded substrates. The RecJ exonucleasehad greater activity on duplex DNA molecules with 5'- ratherthan 3'-single-stranded tails.

The recJ gene of Escherichia coli was initially identified as agene essential for RecBCD-independent pathways for con-jugational recombination and UV repair, the so-called"RecF" and "RecE" pathways (1, 2). In wild-type strains,conjugational recombination proceeds predominantly by theRecBCD pathway and is unaffected by mutations in recJ.However, recJ is required for other types of recombinationalevents, even in RecBCD+ strains. For instance, recombina-tion between plasmids is highly dependent on recJ, as well ason several other RecF pathway genes, and independent ofrecBCD (3). Of the genes for the RecF pathway (recF, recJ,recO, recN, recQ, and ruv), mutations in recJ have the mostextreme effects on recombination, reducing recombinantfrequencies 1000- to 10,000-fold (1-6).

Additional insight into the role of the recJ gene has comefrom analysis of mutants in the recD-encoded subunit of theRecBCD nuclease. Conjugational recombination in recDmutants occurs at slightly elevated frequencies and UV repairappears normal, although the exonuclease activities of theRecBCD enzyme are either absent or greatly altered (7-9).Conjugational recombination and UV repair in recD mutantsstill require the recC gene (and probably recB), as in wild-type strains, but have gained an additional requirement forrecJ (10). One explanation for this requirement for recJ inrecD mutants is that the RecJ protein substitutes for one ormore of the normal activities of the RecBCD enzyme.We have sequenced the recJ gene and, using this sequence,

have developed a strain that overproduces RecJ protein(unpublished data). Induction of recJ was accompanied bythe induction of a nuclease activity that degraded single-stranded DNA. In this communication, we report the puri-fication of this nuclease activity to apparent homogeneity,confirm that it is the product of the recJ gene, and present apartial characterization of its activity.

EXPERIMENTAL PROCEDURESStrains. RDK1893 (sbcB15 endA Apnc-xth gal thi), con-

structed in this laboratory, was used in overproductionexperiments to reduce possible interference by endonucleaseI and exonucleases I and III. RDK1896 is a derivative ofRDK1893 carrying recJ284::TnJO. The construction of recJ-overproducing plasmids will be described in detail elsewhere.Descriptions of the relevant plasmids follow. The pT7-5overexpression vector carries a T7 promoter upstream of thepolylinker site in a derivative ofpBR322 (pT7-5 was providedby S. Tabor and C. Richardson, Harvard Medical School).pGP1-2 (11), also included in each overexpresser strain,carries the gene for T7 RNA polymerase under control of theA PL promoter and the A cI857 repressor gene. pRDK110 ispT7-5 with the EcoRI-Sal I fragment of recJ derived fromplasmid pJC763 (12). pRDK115 is similar to pRDK110 exceptthat a synthetic ribosome-binding site is substituted for thesequences upstream of the initiating codon. pRDK112 is acontrol plasmid that carries the same ribosome-binding site aspRDK115 but is fused to a downstream methionine codonand, therefore, encodes a nonfunctional RecJ protein deletedfor the first 47 amino acids. All overexpression strains aretransformants of RDK1893: RDK1916 contains pGP1-2 andpT7-5, RDK1923 contains pGP1-2 and pRDK110, PDK1918contains pGP1-2 and pRDK115, and RDK1913 containspGP1-2 and pRDK112.Enzymes. Restriction endonucleases were obtained from

New England Biolabs and used as suggested by the manu-facturer. Exonuclease III was purified according to Rogersand Weiss (13). T7 gene 6 exonuclease was purchased fromUnited States Biochemical and reaction conditions werethose specified by the manufacturer.

Nucleic Acids. T7 [3H]DNA was prepared according to theprocedure of Hinkle and Chamberlain (14) using [3H]thymi-dine from New England Nuclear and had a specific activityof 2.4 x 105 cpm/nmol of nucleotide. T7 [3H]DNA withsingle-stranded tails was produced by incubation of T7[3H]DNA with exonuclease III or T7 gene 6 exonucleaseunder conditions that converted 5% of the DNA to trichloro-acetic acid-soluble material; this DNA was further purified byextraction with phenol and by gel filtration on Bio-Gel P-100(Bio-Rad). Mung bean nuclease digestion of these substratesconfirmed that they contained single-stranded regions of thesize predicted by the extent of prior exonuclease digestion.M13mp7 (15) viral and covalently closed, supercoiled repli-cative form (RF) DNA (RFI DNA) was purified from JM101-infected cells as described (16). M13mp7 RF DNA containinga single-stranded interruption (RFII DNA) was produced byincubation ofRFI DNA at 75°C overnight in buffer containing1 M NaCl, 10 mM Tris HCI (pH 8.0), and 1 mM EDTA (17),

Abbreviation: RF DNA, replicative form DNA.*To whom reprint requests should be addressed at the Dana-FarberCancer Institute.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Table 1. Nuclease activity in recJ mutant andrecJ-overproducing strains

Nuclease activity,units/mg of protein

Strain Plasmid ss T7 DNA ds T7 DNA

RDK1893 (rec') 27 4.2RDK1896 (recJ284) 9 4.9RDK1916 pT7-5 (vector) 27 4.8RDK1923 pRDK110 (recJ+) 580 4.0RDK1918 pRDK115 7400 5.6

(RBS/recJ+)RDK1917 pRDK112 14 4.0

(RBS/ArecJ)All strains are derivatives of RDK1893. Crude extracts were

prepared and assayed except that native T7 DNA was substituted fordenatured T7 DNA as indicated. ss, Single stranded; ds, doublestranded.

followed by precipitation with ethanol. Linear virionM13mp7 DNA and linear RF DNA (RFIII DNA) wereprepared by digestion with EcoRI followed by phenol ex-traction and precipitation with ethanol.

Nuclease Assay. Standard reaction mixtures contained 3nmol of T7 [3H]DNA, 20 mM Tris-HCl (pH 8.5), 10 mMMgCl2, 0.67 mM dithiothreitol, and bovine serum albumin (1mg/ml) in 100 ,u. Substrate DNA was heat-denatured byincubation at 100°C for 10 min followed by quenching on ice.If required, protein samples were diluted in a buffer contain-ing 10 mM Tris HCl (pH 7.5), 10mM 2-mercaptoethanol, andbovine serum albumin (0.5 mg/ml). Two microliters of asample or sample dilution was added to the reaction mixtureand the reaction mixtures were incubated at 37°C for 20 min,unless otherwise specified. The reactions were terminated byaddition of 0.3 ml of salmon sperm DNA (0.167 mg/ml) and0.4 ml of 1 M trichloroacetic acid followed by incubation onice for 5 min. The solution was centrifuged for 10 min in aMicrofuge at 4°C and 0.4 ml of the supernatant was removedand added to 4 ml of Aquasol (New England Nuclear)scintillation fluid, and radioactivity was measured in a scin-tillation counter. One unit of nuclease activity is defined asthe amount of nuclease producing 1 nmol of acid-solublenucleotides in 20 min at 37°C.

In some experiments, the reaction products were analyzedby electrophoresis through agarose gels. Reaction conditionswere the same as in the standard exonuclease assay aboveexcept that the total volume was 20 ,ul with 200 ng of eachsubstrate DNA. Reactions were performed in duplicate andcontained either no RecJ protein or 100 ng offraction V. Afterincubation at 37°C for 20 min, 10% (wt/vol) NaDodSO4 wasadded to 0.1%, 0.5 M EDTA was added to 50 mM, andproteinase K (2 mg/ml) was added to 20 Ag/ml, and thereaction mixtures were incubated for an additional 15 min at37°C. At this time, 4 ,ul of 0.25% bromophenol blue, 10 mMEDTA (pH 8.0), and 60% (wt/vol) sucrose was added, andthe samples were heated to 75°C for 2 min and placed on ice.A 5-,lI aliquot of this was loaded on a 1% agarose gel in buffercontaining 40 mM Tris borate (pH 7.9) and 1 mM EDTA and

subjected to electrophoresis for 2 hr at 75 V. The gel was

stained with ethidium bromide (1 ,ug/ml) for 30 min andphotographed. The photograph was then scanned with an

LKB Ultrascan densitometer to determine the amount ofDNA present in individual bands.Crude Extracts. Plasmid-containing strains were grown

with shaking at 30°C in 2YT medium (15) containing 100 mMpotassium phosphate (pH 7.0), 0.2% glycerol, kanamycin (30,g/ml), and ampicillin (100 ,ug/ml). After the culture hadreached an OD590 of 0.8, cells were induced by shaking at42°C for 60 min. Rifampicin was then added to 100 ,ug/ml toinhibit expression from E. coli promoters and permit exclu-sive expression from the T7 promoter on the expressionplasmid. Then the cultures were incubated for an additional15 min at 42°C and then switched to 37°C for 1 hr beforeharvesting. Non-plasmid-containing strains were grown inLB medium at 37°C until the culture attained an OD590 of 0.8.Cells were harvested by centrifugation in a Sorvall GS3 rotorat 7000 rpm for 10 min at 4°C, washed with 0.1 vol of buffercontaining 50 mM Tris-HCl (pH 7.5) and 10% (wt/vol)sucrose and resuspended in 0.01 vol of the same buffer. Thecell suspensions were then frozen in liquid nitrogen andstored at -70°C. Frozen cells were thawed at room temper-ature and placed on ice when thawing was completed. To thethawed cell suspension were added 5 M NaCl plus lysozyme(10 mg/ml) to final concentrations of 0.2 M and 0.2 mg/ml,respectively. The cell suspension was incubated on ice for 45min, then heated with a 37°C water bath until the temperatureof the suspension reached 20°C, at which time it was returnedto ice until the temperature reached at least 10°C. Small-scalecrude extracts (which were used to determine relative nu-clease activity) were then centrifuged 15 min in a Microfugeat 4°C and the supernatant was assayed as described above.Values presented for these crude extract assays are repre-sentative of two or more determinations. Large-scale crudeextracts were centrifuged at 40,000 rpm in a Beckman 60 Tirotor at 4°C for 20 min.RecJ Exonuclease Purification. All steps were performed at

4°C unless otherwise stated. The standard buffer (buffer A)throughout the purification contained 20 mM Tris HCl (pH7.5), 10% (wt/vol) glycerol, 0.1 mM EDTA, and 1 mMdithiothreitol; various concentrations of NaCl or ammoniumsulfate were added as indicated. All chromatography col-umns had a cross-sectional area of 0.64 cm2 and were run ata flow rate of 6-8 ml/hr. A 2-liter culture of RDK1918(RDK1893 transformed with pGP1-2 and pRDK115) wasgrown, harvested, and stored frozen as described above. Alarge-scale crude extract (fraction I, 20 ml) was prepared. Tothis crude extract, 7.4 g of ammonium sulfate was added withstirring over a 20-min period. After stirring for an additional15 min, the precipitate was collected by centrifugation in aSorvall SS34 rotor at 15,000 rpm for 10 min and resuspendedin 26 ml of buffer A to yield a protein solution having aconductivity equivalent to buffer A containing 100 mM NaCl(fraction II). Fraction II was applied to a 4-ml column ofPBE94 (Pharmacia) equilibrated with buffer A containing 100mMNaCl. The column was washed with 16 ml of buffer Acontaining 100 mM NaCl and the activity was eluted with a

Table 2. Purification of the single-stranded-DNA-specific nuclease froma recJ-overproducing strain

Protein, Activity, Specific activity, Yield,Fraction mg units units/mg

I. Crude extract 50 420,000 8,400 100II. Ammonium sulfate 37 470,000 13,000 110III. PBE 94 8.1 310,000 38,000 74IV. Blue agarose 0.46 170,000 370,000 40V. Hydroxylapatite 0.20 59,000 300,000 14RecJ protein was purified from 2 liters of induced RDK1918 cells.

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100-ml linear gradient from 100 mM NaCi to 1 M NaCi inbuffer A. The peak of activity was eluted at =230 mM NaCi.The active fractions were pooled (fraction III, 7.5 ml) anddiluted with buffer A to yield 16.5 ml of a protein solutionhaving a conductivity equivalent to buffer A containing 100mM NaCi. This solution was applied to a 3-ml column ofCibracron Blue 3GA-agarose (type 100, Sigma) that had beenequilibrated in buffer A containing 100 mM NaCi. Afterwashing the column with 10 ml of the same buffer, a 75-mllinear gradient from 100 mM to 1 M NaCi in buffer A wasapplied. The activity was eluted in a broad peak at a NaCiconcentration centering around 210 mM NaCi and the activefractions were pooled (fraction IV, 24 ml). Fraction IV wasdialyzed against 2 liters of buffer A containing 250 mM NaCiand applied to a 1-ml hydroxylapatite (Bio-Gel HTP, Bio-Rad) column equilibrated with buffer A containing 250 mMNaCi. After washing with 4 ml of the same buffer, a 50-mllinear gradient from buffer A containing 250 mM NaCi tobuffer A containing 1 M ammonium sulfate was applied. Theactive fractions were eluted at a salt concentration having aconductivity equivalent to buffer A containing 230 mMammonium sulfate. The fractions containing activity werepooled (5.5 ml), dialyzed overnight against a 1-liter solutioncontaining 50 mM Tris-HCl (pH 7.5), 60% (wt/vol) glycerol,0.1 mM EDTA, and 1 mM dithiothreitol and stored at -200C(fraction V).

Protein Analysis. Protein concentrations were determinedby the method of Bradford (18) using the standard reagentpurchased from Bio-Rad and bovine serum albumin asstandard. Protein samples were analyzed by NaDodSO4/PAGE (19) using a Mighty Small II apparatus (Hoefer) with0.75-mm gels and staining with Coomassie blue R-250. Forprotein sequencing, 10 tg of fraction V was precipitated with10 vol of ethanol at -20'C. The N-terminal sequence of thismaterial was determined by Ruth Steinbrich (Laboratory ofImmunobiology, Dana-Farber Cancer Institute) using anApplied Biosystems model 470A sequencer equipped with amodel 120A on-line phenylthiohydantoin analyzer.

Renaturation After NaDodSO/PAGE. For the renaturationexperiment, 200 units (%1 ,ug) of fraction V was loaded induplicate on a 0.75-mm-thick NaDodSO4/polyacrylamide gel(19) with prestained protein markers (Sigma) in an adjacentwell. After electrophoresis for 1.5 hr at 20 mA, the gel wassliced into duplicate halves. One half of the gel was stainedwith Coomassie blue for reference and the other half wasgently shaken with 50 ml of buffer A containing 150mM NaCIfor 90 min at 4°C with a buffer change after each 30-minperiod. At this point, the prestained protein markers werestill visible although the bromophenol blue dye had justemerged from the gel. The gel was then cut into 2.5-mmslices, each slice was added to 100 ,ul of buffer containing 10mM Tris-HCl (pH 7.5), 10 mM 2-mercaptoethanol, andbovine serum albumin (0.5 mg/ml), crushed with a pestle,and incubated for 72 hr at 4°C. Each fraction was thenassayed for nuclease activity using single-stranded DNAsubstrates as described above.

RESULTSIdentification and Purification of RecJ Nuclease. Assays of

crude extracts of wild-type and recJ mutant strains and recJ-and mutant recJ-overproducing strains revealed that a nu-clease that specifically degraded single-stranded DNA wasassociated with the recJ gene (Table 1). This nuclease activitywas reduced in a recJ mutant (RDK1896) relative to a recJ+strain (RDK1893). Induction of an isogenic strain carrying aplasmid with recJ under the control of a heat-induciblepromoter (RDK1923) increased this activity 20-fold; additionof a more optimal ribosome-binding site upstream of thecloned recJ gene (strain RDK1918) gave a 270-fold increase

a b c d e f

66,000- vrm_

43,000- via

FIG. 1. NaDodSO4/PAGE analysis of various RecJ proteinfractions. Crude extracts and purified protein fractions were pre-pared. Lanes: a, crude extract (19 ug) of RDK1916 (non-RecJ-overproducer); b-f, fractions I-V of nuclease purified from RecJ-overproducer RDK1918 as described in Table 2. The amount ofprotein loaded per well corresponds to 4150 units of nucleaseactivity. Lanes: b, 19 gg of fraction I; c, 12 ,ug of fraction II; d, 4 Agof fraction III; e, 0.4 ,ug of fraction IV; f, 0.5 ,ug of fraction V. Themarkers used included lysozyme (14.4 kDa), soybean trypsin inhib-itor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (42.7 kDa),bovine serum albumin (66.2 kDa), phosphorylase b (97.4 kDa),f3-galactosidase (116.25 kDa), and myosin (200 kDa) and werepurchased from Bio-Rad.

of this nuclease activity relative to strains carrying noplasmid (RDK1893) or vector alone (RDK1916). Induction ofa control plasmid that overproduces a noncomplementingtruncated RecJ protein (RDK1917) yielded extracts havingslightly less nuclease activity than extracts of RDK1916 orRDK1893, both of which lack an overproducing plasmid. Nodifference in nuclease activity was seen among these strainswhen double-stranded DNA was used as a substrate.The nuclease activity induced in strain RDK1918 was

purified to homogeneity and the purification steps are sum-marized in Table 2 and Fig. 1. Fractions IV and V containeda single 60-kDa polypeptide that was induced by recJ over-expression. This protein was not observed in a crude extractof the control strain carrying the vector plasmid lacking therecJ gene (Fig. 1). The purity of fraction V was >98%, asestimated by NaDodSO4/PAGE (Figs. 1 and 2). Proceduresfor specifically labeling the plasmid-encoded proteins [both"maxi-cell" (12, 20) and post-rifampicin labelings (11)] re-

A. (top)

C

c

Co

co0)

-o

0)LG

(bottom)

1 2 3 4 5 6 7 8 9 10111213141516

Fraction number

B.

FIG. 2. Renaturation of RecJ nuclease after fractionation byelectrophoresis through a NaDodSO4/polyacrylamide gel. FractionV (1 Ag; 200 units) was fractionated in duplicate by NaDodSO4/PAGE. (A) One lane was renatured and cut into slices, and theprotein was eluted. A 2-pLI aliquot of each fraction was assayed fornuclease activity on single-stranded-DNA substrates. Twelve per-cent of the applied activity was recovered. (B) The second lane wasstained with Coomassie blue and served as a marker for the RecJprotein.

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2630 Biochemistry: Lovett and Kolodner

Met Lys Gln Gln Ile His Leu Arg Arg Arg Glu Val Asp Glu Thr Ala Asp Leu

GTG AAA CAA CAG ATA CAC CTT CGT CGC CGT GAA GTC GAT GAA ACG GCA GAC TTG

FIG. 3. Comparison of the RecJ protein sequence with the RecJ DNA sequence. The first 18 amino acids determined by sequencing fractionV are displayed by aligning with the first 54 nucleotides of RecJ DNA coding sequence starting with the initiation codon. The complete sequence

of the RecJ region of E. coli will be presented elsewhere.

sulted in the labeling of a 60-kDa polypeptide that comi-grated with this protein (data not shown).That this polypeptide is the product of the recJ gene and is

the nuclease was confirmed by two experiments. (i) Frac-tionation of this protein by NaDodSO4/PAGE followed byrenaturation and nuclease assays of these fractions showedthat all the single-strand-specific nuclease activity comi-grated with the 60-kDa protein, the only protein visible on thegel (Fig. 2). (ii) Eighteen cycles of N-terminal sequenceanalysis ofthe protein present in fraction V gave the 18 aminoacids predicted from the nucleotide sequence of the recJgene, as illustrated in Fig. 3.

Characterization of RecJ Nuclease Activity. A time courseof digestion of single-stranded DNA by various amounts ofRecJ protein is presented in Fig. 4. The reaction was linearonly at early time points containing low concentrations ofRecJ protein. At the maximal rate, 1 molecule ofRecJ proteindigested 1000 nucleotides per min. Addition oflarge amountsof RecJ protein resulted in digestion of 90o of the substrateDNA. The pH optimum of the reaction was between 8.0 and8.5, close to our standard reaction conditions (data notshown). The reaction required Mg2" and the sulfhydrylreducing agent dithiothreitol but did not require ATP (Table3). Mn2' not only was unable to substitute for Mg2' but alsoinhibited the enzyme in the presence of Mg2+. Cu2*, Fe2+,Co2+, Ca2+, and Zn2+ behaved similarly to Mn2+ (data notshown).No nuclease activity was detected on double-stranded T7

DNA substrates under standard reaction conditions with theamount of acid-soluble material observed the same as thecontrol with no added enzyme (Table 4). No double-strand-specific exonuclease activity was detected whenlarger amounts of RecJ protein were assayed. The highestlevel of double-strand-specific exonuclease activity that frac-tion V could have was <30 units/mg, which was 10,000-foldless than the activity on single-stranded DNA. Likewise, theaddition of a 10-fold excess of unlabeled double-stranded T7DNA did not inhibit the enzyme's activity on single-strandedDNA (data not shown). Under conditions that result in 30%digestion of an equivalent amount of denatured T7 DNA, T7

100'1

80

0 60

01

0 20 40

Minutes of incubation

FIG. 4. Time course of degradation of denatured T7 DNA. Theamounts of RecJ protein (fraction V) present in each reaction are as

indicated.

DNA containing 5'-single-stranded overhangs was digestedto an extent corresponding to 70% of the available single-stranded tail (Table 4). T7 DNA containing 3'-single-strandedtails was degraded to a much lower extent, suggesting thatRecJ protein preferentially initiates degradation at 5' ratherthan 3' ends. This has been confirmed in experiments using5'- and 3'-end-labeled substrates (unpublished results).The ability of RecJ protein to degrade various forms of

M13mp7 virion and RF DNA was also analyzed by agarosegel electrophoresis. RecJ protein only digested linear single-stranded DNA and had little or no effect on supercoiled,nicked circular, or linear double-stranded DNA or on circularsingle-stranded DNA. From the densitometer tracings of thegel photographs, we estimate that the maximum amount ofeither double-stranded or single-stranded DNA endonucle-ase activity present in fraction V was <0.05 nmol of DNAmolecules cleaved per mg of protein. This amount of activityis 21,000,000 times less than the single-stranded-DNA-specific exonuclease activity. Identical results were obtainedwhen 1 mM ATP was included in these reactions (data notshown). The sum of these results indicates that RecJ proteinis a single-stranded-DNA-specific exonuclease.

DISCUSSIONWe have detected a single-stranded-DNA-specific nucleasethat is encoded by the recJ gene and have purified it toapparent homogeneity from a RecJ-overproducing strain.The nuclease requires a free single-stranded end and has nodetectable activity on double-stranded DNA. It appears topreferentially degrade single-stranded DNA from the 5' end.However, further analysis will be required to determine theextent, if any, of its ability to degrade single-stranded DNAfrom the 3' end. These properties distinguish the RecJ proteinfrom the two major single-stranded-DNA-specific exonu-cleases in E. coli, exonuclease I and exonuclease VII. RecJprotein was purified from an exonuclease I-deficient mutantand differs in apparent polarity of digestion of single-strandedDNA compared to exonuclease I (21). Exonuclease VIIdegrades single-stranded DNA in both directions, has adifferent size compared to RecJ, and does not require addeddivalent cations for activity (22). Further analysis of themolecular structure of RecJ, its processivity, polarity ofdegradation, substrate specificity, and reaction products will

Table 3. Reaction requirementsAlteration(s) to the complete

reaction mixture Relative activity, %

None 100- Enzyme 1- mg2+ 1- Dithiothreitol 4- Bovine serum albumin 110- Mg2+ + Mn2+ (10 mM) 3+ Mn2+ (10 mM) 5+ ATP (100 jIM) 100+ NaCl (100 mM) 110

Reactions contained 2 ng of RecJ protein (fraction V) and 3 nmolof heat-denatured T7 DNA (as nucleotides). Incubation was for 10min at 370C. The 100% relative activity corresponds to 0.3 nmol ofacid-soluble nucleotides.

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Table 4. Substrate requirementsSubstrate Digestion, %

T7 DNA 0.2*Heat-denatured T7 DNA 303'-single-stranded-tailed T7 DNA 0.75'-single-stranded-tailed T7 DNA 3.4

Reaction mixtures contained 20 ng ofRecJ protein (fraction V) and3 nmol of DNA. Incubation was for 10 min at 370C. Control reactionmixtures with no added enzyme gave 0.2% of the acid solubility ofeach substrate and this value was not subtracted from the givenvalues. Tailed DNA was produced by digestion to 5% of acidsolubility with T7 gene 6 exonuclease (3' tailed) or exonuclease III(5' tailed). In both cases, the tails corresponded to 5% of the totalmolecule.*Identical to the control with no added enzyme.

be essential to understand the function of recJ in recombi-nation in vivo.Although the exact role that RecJ protein plays in recom-

bination and repair is still unclear, its biochemical propertiessuggest several possible functions. For instance, RecJ pro-tein may be required to process single-stranded intermediatesthat are formed during recombination. Examples of suchintermediates are those formed when RecA protein pairshomologous linear double-stranded DNA with DNA mole-cules containing single-stranded regions (23, 24). In addition,RecJ protein could act in conjunction with proteins such ashelicases to produce single-stranded gaps and 3'-single-stranded ends in duplex DNA molecules, structures that arethought to be substrates for recombination. This latter idea isconsistent with observations that the presence of exonu-clease I causes inhibition of recJ-dependent recombination incertain strains (2, 25).The interplay between various nucleases appears to be

crucial for recombination and repair. recJ-dependent recom-bination substitutes effectively for RecBCD-dependent con-jugational recombination and UV repair in recD mutants (10)or if either exonuclease I (3' to 5', single-stranded-DNAspecific) is eliminated by sbcB mutations (2, 20, 25, 26) orexonuclease VIII (5' to 3', double-stranded-DNA specific) isinduced by sbcA mutations in recB recC mutants (27-29).This may reflect a complex and sometimes competitiveprocessing ofDNA structures critical for recombination andrepair by these various nucleases. However, it remainspossible that these proteins have recombinational activitiesother than their nuclease activities. Further analysis will berequired to understand the role of these enzymes in recom-bination and repair.We thank Drs. Ruth Steinbrich and Ellis Reinherz for performing

the N-terminal sequence analysis and Drs. David Norris and ThomasGriffin IV for helpful comments on the manuscript. This work wassupported by Grant GM26017 from the National Institutes of Healthand Faculty Research Award FRA-271 from the American CancerSociety to R.D.K.

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