Isolation of Altered recA Polypeptides and Interaction with ATP and

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 2, Issue of January 25, $P: 949-955,1985 rrnted m U.S.A.

Isolation of Altered recA Polypeptides and Interaction with ATP and DNA*

(Received for publication, June 18, 1984)

James R. RuscheS, William Konigsberg, and Paul Howard-Flanders From the Department of Molecular Biophysics and Biochemistry, and Department of Therapeutic Radiology, Yale University, New Hauen,Connecticut 0651 1

In this paper we describe the partial proteolytic digestion of recA proteins from Escherichia coli and Proteus mirabilis and the production and isolation of truncated recA polypeptides. A proteolytic fragment of the P. mirabilis recA protein bound single-strand DNA and ATP normally but has altered duplex DNA binding properties. This protein was shown to initiate but not complete DNA strand transfer from a DNA duplex to a complementary single strand. The product of the E. coli recAl allele bound but could not hydrolyze ATP and the protein bound single-strand but not double-strand DNA. This protein did not appear to initiate the transfer of a strand from a linear duplex to a single- strand circle and inhibited the wild-type recA protein from performing strand transfer.

We report that recA protein binds linear duplex DNA in a manner that enhances the rate of ligation by T4 DNA ligase. When heterologous single-strand DNA was added in addition to the duplex DNA large stable aggregates of protein and DNA were formed that could easily be sedimented from solution.

recA protein is directly involved in the regulation of indu- cible DNA repair and the enzymology of DNA recombination (for review, see Refs. 1 and 2). Although of modest size (37,800 daltons), recA protein demonstrates single-strand DNA binding (3), DNA-dependent ATPase (4, 5), DNA- and ATP- dependent stimulation of proteolytic cleavage of lexA and X CI proteins (5), double-strand DNA binding (6, 7), self-aggregation to form filaments either free in solution (6) or on DNA (8), and transfer or exchange of duplex DNA pairing (9, 10). These studies suggest that several active sites exist within this protein. recA proteins from various bacteria share some structural and functional properties since recA-like proteins show antigenic relatedness (11,12) and recA genes transferred between bacterial species have been active (13,14). We would like to understand how these active sites function in concert to produce protein-DNA complexes that facilitate DNA recombination.

In this paper we describe the isolation and characterization of altered recA polypeptides and a comparison by partial proteolytic digestion of the recA proteins from Escherichia coli (recAec) and Proteus mirubilis (recApm). We examined the interaction of recA polypeptides with ATP and DNA and found the product of the E. coli recAl allele can bind but not

CA 20763 and CA 09259-06. The costs of publication of this article * This work was supported by National Institutes of Health Grants

were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Fellow of the American Cancer Society.

hydrolyze ATP. This mutant protein bound single but not double-strand DNA and inhibited the wild-type protein from catalyzing strand transfer. A proteolytic fragment of the P. mirabilis recA protein binds single-strand DNA and ATP normally but has altered duplex DNA binding properties. This polypeptide can initiate but not complete the transfer of a DNA strand from a linear duplex to a single-strand circular DNA molecule.

We also report that recA protein can bind duplex DNA in a manner that enhances the rate of DNA fragment ligation by T4 DNA ligase. In the presence of single strand and duplex DNA, recA protein forms large protein-DNA aggregates although the DNA molecules share no homology.

EXPERIMENTAL PROCEDURES

Bacteria and Plasmids-E. coli strain CGSC 6172 (Alac-pro, F’lacIqL8) was obtained from Dr. B. Bachmann, Yale University, and the strain harboring the recAl (15) allele was from Dr. J. Clark, University of California, Berkeley. pACYC184 (16) and pBR322 (17) were maintained in E. coli strain AB2487 (thyA, recAl3). Plasmid pTL12 was a gift from Dr. Tsung-chung Lin, Yale University; this is a pUC9 plasmid (18) containing the TAC 12 promoter (19). Plasmid pMH21 (20) was a gift from Dr. S. Sedgwick, National Institute for Medical Research, Great Britain. This plasmid was constructed by inserting a BamHI fragment, containing an E. coli recA gene truncated by insertion of a yb element, into pBR322. The 2-kilobase pair fragment contains the recA gene transcription control signals, 60% of the recA coding sequence terminating at a translational stop site located within the yb element. This allele is designated recAl6. For overproduction of this truncated gene product we inserted the BamHI fragment into pTL12, creating plasmid pJR202, and CGSC 6172 cells harboring this plasmid produced large amounts of a 26,000-dalton protein (60% of recA protein) upon addition of isopropylthiogalacto- pyranoside, an inducer of the TAC promoter.

Preparation of DNA-The methods used for preparing DNA have been previously described for plasmid DNA (21), 6X174am3 phage DNA and RF I DNA (22). Full length linear duplex molecules were produced from form I DNA by restriction enzymes using reaction conditions prescribed by the manufacturer. All DNA was dialyzed to equilibrium against 10 mM Tris-HC1, pH 7.5, 0.5 mM EDTA prior to use. DNA concentrations are expressed as moles of nucleotides.

Proteins-The nomenclature of mutant recA proteins used in this study is meant to indicate the species of origin and provide an identifying number which refers to either a genetic allele that codes for the protein or the protein molecular weight. Thus the product of the recAl allele of E. coli is called the recAecl protein and a proteolytic fragment of 36,000 daltons cleaved from the P. mirabilis recA protein is termed the recApm36 protein. Purified recAec (22) and recAec1 (8) were kindly provided by Dr. S. West, Yale University, and recApm was prepared as described previously (11).

To prepare the truncated protein recAec26, cells harboring pJR202 (1.2 liters) were grown to late log phase and isopropylthiogalactopy- ranoside added to 1 mM. After 6 h the cells were collected and stored at -80 “C in R buffer (20 mM Tris-HC1, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride) at 2 ml/g. A cleared lysate (28 ml) was prepared as described (22) and protein purification was monitored by electrophoresis of aliquots on

949

950 Functional Organization of recA Protein

polyacrylamide gels containing SDS.’ The recAec26 protein was polyacrylamide gel. While a large fragment of about 36,000 precipitated by the addition of PolYmin p to 0.5%- The Pellet Was daltons was produced from both proteins by subtilisin (Fig. 1, washed twice with R buffer containing 100 mM (NHdzSO, (8 ml). lane uerSuS 5 ) only the r e c ~ p m fragment is stable enough The resulting supernatants were pooled and made 50% in (NH&SO,. The protein pellet was resuspended and dialyzed against R buffer. to be Observed as a major product Of partial proteolysis- v8 The dialysate was applied to an Affi-Gel blue column (25 ml) and the Protease, which cleaves peptide bonds at the carboxyl-termi- bound protein eluted with a 0-1 M KC1 gradient. Peak fractions were nal side of either aspartic or glutamic acid (27), produced pooled, dialyzed versus R buffer, and applied to a DEAE-cellulose protein fragments ranging from 7 to 32 kDa. Major fragments column (9 ml). Adsorbed protein was eluted with a 10-column volume from v8 digestion were to both r e c ~ pro- gradient containing 0-0.25 M KC1 in R buffer. Peak fractions were teins (Fig. 1, lane UerSLIS made 50% in ammonium sulfate and the resulting protein precipitate was resuspended and dialyzed versus R buffer containing 20% glyc- Production and Purifkation Of recA Protein Frqmnts- erol. Since subtilisin digestion of recApm produces a large stable

Double diffusion immunoprecipitation in agar was performed using protein fragment, we purified this polypeptide by gel filtration immune sera raised against the intact recAec protein (11). A single (see ‘‘Experimental Procedures”). The fragment had an ap- precipitin band developed between antibody and recAec25. This line parent molecular weight of 36,000 on denaturing polyacryl- of precipitated protein showed partial identity with a precipitin band amide gels (Fig. 5, lane 5 ) and thus will be referred to as formed between intact recAec and the antisera ( d a t a not shown).

Purification of a 36,000-dalton recApm fragment generated by recApm36* The amino-termina1 sequence Of recApm and subtilisin digestion was routinely performed in a reaction mixture recApm36 was determined (see “Experimental Procedures”) (2.0 ml) containing 3.0 mg of recApm, 1 pg of subtilisin, 12 mM Tris- and both proteins have the amino-terminal sequence NH,- HCl, pH 7.5, and 4% glycerol. After incubation at 37 “C for 1 h, Ala-Ile-Asp-Glu. This is the Same amino-terminal sequence phenylmethylsulfonyl fluoride Was added to 5 mM and the Sample as reAec (25). Thus subtilisin removed about 25 amino layered on a Bio-Gel A-0.5m column (28 X 1.6 cm) and 1-ml fractions were collected. The excluded and included volumes of the gel filtration from the terminus Of recApm* column were determined with [3H]DNA of ,$X174 phage and 3zpi, We also Purified a 26,000-dalton Protein coded for by a respectively. The excluded material was precipitated with 50% am- truncated E. coli recA gene (20; see “Experimental Proce- monium sulfate and resuspended at 1.0 mg/ml. This material was dures”). After subcloning the truncated gene to an expression free of intact recApm as determined by SDS-gel electrophoresis (see vector, plasmid-bearing cells Were induced and the recAec26 Fig. 5, lane 5 ) and was used in all experiments.

Assays-ATPase assays were performed as described previously polypeptide was purified as described under “Experimental (8). A reaction mixture (20 pl) containing 80 m~ Tris-HCl, pH 7.5, Procedures”’ A denaturing polyacrylamide gel shows each step 3.3 mM MgClz, 3.3 mM 2-mercaptoethanol, 0.1 mg/ml, bovine serum in the purification (Fig- 2). albumin, 0.25 mM [c~-~’P]ATP (4000 cpm/nmol), and 12 p~ ssDNA Protein was purified from the E. coli mutant r e d l (15). was incubated at 37 “ c . 2 were spotted onto polyethyleneimine The recAecl allele has a missense mutation at amino acid 160 thin-layer chromatography Plates and chromatographed in 0.75 M which replaces a glycine with an aspartic acid residue (28). counted and the per cent ADP calculated. Background ADP levels were about 5%. and recAecl allowed us to compare them with wild-type recA

EDTA, 0.75% SDS, 0.01% bromphenol blue and appliedto 1% agarose Interaction with ATP-Since recA protein is an ATPase gels. After electrophoresis at 90 V for 4 h, gels were dried and exposed (4, 5) we examined the altered proteins for the ability to bind to XAR-5 film (Kodak). Electrophoresis of proteins in the presence ATP. ~ i ~ d i ~ ~ to wild-type r e c ~ was demonstrated by the of SDS was according to Laemmli (23) in discontinuous polyacrylamide gels. Protein bands were visualized by staining with Coomassie covalent linkage of the photoactivatable analogue 8-azido- [a- Blue R-250 (Bio-Rad) or radiographic exposure of dried gels. 32P]ATP to the protein followed by SDS-gel electrophoresis

Protein sequencing from the amino terminus by the solid phase (Fig. 3A). Labeling required UV light activation and unlabeled method (24) was kindly performed by Dr. K. Williams, Yale Univer- ATP reduced the amount of3,p co-migrating with recAec(85% sity. The first four amino acids OfrecApm, recApm36, and the control reduction, data not shown). recApm36 and recAec1 were also recAec (25) were determined. All three polypeptides had the identical labeled by 8-azido-ATp in a manner sensitive to sequence: NHz-Ala-Ile-Asp-Glu.

Krasnow and Cozzmelli (26). Reaction mixtures were incubated as azido-ATP was inefficient and no decrease in the amount of indicated and immediately placed in a microcentrifuge and spun for associated radioactivity was seen with the addition of unla- 30 8 (15,000 x g). A portion of the supernatant was counted by liquid beled ATP ( d a t a not shown). Therefore while recApm36 and scintillation.

Materials-Phosphocreatine, phosphocreatine kinase, isopropyl- recAecl bind ATP, recAec26 does not appear to have a

thiogalactopyranoside, proteases V8, and subtilisin were purchased functional ATP binding site.

DEAE-cellulose was from Whatman and Betafluor was from National polypeptides to hydrolyze ATP to ADP in the presence of Diagnostics. 8-Azid0-5’-[(u-~~P]ATP was obtained from ICN. T4 li- single-stranded DNA. Fig. 4, top, shows that recApm36 effi- gase and type 11 restriction enzymes were purchased from New ciently hydrolyzed ATP whereas recAec1 did not demonstrate England Biolabs. any ATPase activity under standard reaction conditions. The

initial rate of ATP hydrolysis catalyzed by recApm and recApm36 was examined by observing a time course of ADP

proteolytic Digestion of r e c ~ proteim from E. coli and p. formation. Both the rate and extent of ATP hydrolysis were mirabil~-previous studies in (13) and in vitro (11, 29) identical when incubating equimolar amounts of the proteins have shown the functional similarity between recAec and (Fig. 4, bottom). As with the intact Protein, recApm36 required recApm proteins. We compared the structural organization of the presence of single-stranded DNA in order to catalyze ATP these proteins by partial proteolytic digestion. recAec and hydrolysis. recApm were treated with either subtilisin or V8 protease and recAPm-catalYzed C k a u W o f x RePressor-previous studies the resulting polypeptide fragments displayed on a denaturing have demonstrated that recAec1 does not cleave repressor

under conditions optimum for the wild-type protein (4). Re- t The abbreviations used are: SDS, sodium dodecyl sulfate; ssDNA, cently, West and Little (29) have demonstrated that Purified

single strand DNA; ssb, single-strand binding protein. recApm protein will catalyze the cleavage of E. coli lexA and

KPO4, pH 3.5. Radioactivity at the position of ATP and were The purification of the altered peptides recApm36,

Ekctrophresi-Samples containing DNA were made 50 mM proteins in studies on reactions with ATP and DNA.

The aggregation of DNA was measured essentially as described by by nonradioactive ATP (Fig. 3B). Linkage ofrecAec26 and 8-

from Sigma. Bio-Gel A-0.5m and Affi-Gel blue were from Bio-Rad. HYdrolYs~ of ATP-we next examined the ability of r e d

RESULTS

Functional Organization of recA Protein

1 2 3 4 5 6 7 8 9 1 0 1 1 - Origin

951

- 43

- 17.2 - 14.3

0 0.1 1.0 0 0.1 1.0 0.02 0.1 0.02 0. I

Subtilisin (pg) V 8 (pa) FIG. 1. Comparison of proteolytic digestion patterns of recAec and recApm. Reaction mixtures (20 pl)

containing 30 pg of recA protein, 12 mM Tris-HC1, pH 7.5, 4% glycerol (0.05% SDS for V8 protease) and the indicated amount of protease were incubated at 37 “C for 1 h. The mixture was made 5 mM in phenylmethylsulfonyl fluoride and 10 p1 were loaded to a 15% polyacrylamide gel containing SDS. The gel was stained with Coomassie Blue R-250. Lane 7 contains the following molecular weight markers: ovalbumin, 43,000; X cI, 26,500; myoglobin, 17,200; lysozyme, 14,300 Lanes 1,2,3,8, and 9 contain recApm; lanes 4,5, 6, 10, and 11 contain recAec.

1 2 3 4 5 A B - Origin 1 2 3 4 1 2 3 4 5

- Origin -

- 43 - 37.0

\ ret A

recAec - fragment

- 26.5 m

t 8 FIG. 3. Covalent linkage of 8-azido-[~-~~P]ATP to recA pro- .- -0 teins. Reaction mixtures (10 pl) containing 20 mM Tris-HC1, pH 7.5, x 2 mM MgC12, I mg/ml bovine serum albumin, 40 pM 8-azido-ATP,

and 20 PM recA protein were irradiated with 254 nm light for 30 s.

SDS. Gels were dried and exposed to XAR-5 film. A: lane 1, recAec;

1, recAec; 2, recApm36; 3, recApm36 + 100 p~ ATP; 4, recAecl; 5, recAecl + 100 PM ATP.

- 17.2 Aliquots were electrophoresed on 12% polyacrylamide gels containing

- 14.3 2, recAec no UV light; 3, recAec + 100 p~ A T P 4, recApm. B: lane

FIG. 2. Purification of a truncated recAec gene product. Cells harboring pJR202 were induced with 1 mM isopropylthiogalac- topyranoside for 6 h. Details of the purification are given under “Experimental Procedures.” Samples of each stage in the purification were examined by electrophoresis through a 15% SDS-polyacrylamide gel. Lane 1, crude extract, 60 pg; 2, ammonium sulfate, 60 pg; 3, Affi- Gel blue, 30 pg; 4, DEAE-cellulose, 25 pg; 5, same standards as Fig. 1, lane 7 with addition of recAec (37,842 daltons).

X CI proteins in vitro. Therefore we examined recApm36 for the ability to catalyze cleavage of X repressor in the presence of single-strand DNA and ATPyS. Repressor cleavage in the presence of recApm36 produced the same size products (R1 and R2) as the intact protein (Fig. 5).

Binding of Protein to Single-strand DNA-Binding to single-strand DNA was observed by measuring protein-dependent retention of 4x174 phage [3H]DNA on a nitrocellulose filter. Fig. 6 shows that recAec1 and recApm36 retained ssDNA on filters with the same stoichiometry as wild-type proteins. recAec26 did not retain any DNA on the filter. Wabiko et al. (30) examined the filter retention of ssDNA by recAecl and also conclude that in the absence of ATP the recAecl protein bound ssDNA normally.

Protein Binding to Duplex DNA-Little information exists concerning the interaction of recA protein with duplex DNA under physiologic conditions of pH and temperature. There- fore, we wanted to investigate the binding of protein to duplex DNA. Protein-dependent retention of duplex DNA on nitrocellulose filters was assayed as described for single-strand


I 2 3 4 5 6 - Origin

I- z W V (L

PROTEIN (prnol)

0 2 5 10 TIME (rnin)

FIG. 4. ATPase activity of recA proteins. A description of reaction mixtures and method for quantitating ADP formation are described under “Experimental Procedures.” In part A , background levels of ADP (5%) were subtracted from all data points. A: 0, recApm36; 0, recAecl. B: 0, recApm36; 0, recApm; X, recApm36 without ssDNA.

DNA binding. recAec and recApm proteins can retain duplex DNA on filters with a ratio of bound DNA to protein of about 5 base pairs/protein monomer (Fig. 7, B and D). The optimum magnesium concentration was found to be 10 mM with 50% efficiency at 4 and 18 mM (data not shown). recAec1 did not retain duplex DNA (Fig. 7A) even when added a t a ratio of 2 protein monomers/nucleotide. Thus, recAecl appears to bind single-strand DNA with the same stoichiometry as wild-type protein but cannot form a stable complex with duplex DNA.

recApm36 bound duplex DNA efficiently but we observed two differences in this binding from the intact recApm protein (Fig. 7C). At low protein concentrations, recApm (as well as recAec) showed a nonlinear relationship between protein added and DNA retained on the filter. In contrast, the rec- Apm36 protein demonstrated a linear binding profile that reproducibly differed from the intact protein. Second, addition of ATP to the reaction mixture caused a dramatic decrease in the DNA bound by wild-type proteins while DNA retention by the recApm36 fragment was only slightly affected by ATP (Fig. 7C). These two differences indicate that recApm36 binds more tightly to duplex DNA than the intact protein.

Protein-dependent Aggregation of Duplex DNA-Another method employed to examine protein interaction with duplex DNA was to observe formation of protein-DNA aggregates. The assay measures formation of aggregates large enough to be sedimented out of solution by a short, low-speed centrif- ugation (26). Fig. 8A shows that when single-strand [3H]DNA

-0 - roc Apm - - - recApm 36

- RI - R 2

FIG. 5. Cleavage of X repressor by recApm proteins. A reaction mixture containing 20 mM Tris-HC1, pH 7.5, 2 mM MgC12, 1 mM ATP[S], 15 p~ single-strand DNA, 5 pg of recApm protein, and 4 pg of repressor was incubated overnight at 37 “C and electrophoresed through a 13% polyacrylamide gel containing SDS. The gel was stained with Coomassie Blue R-250. Lanes 1 and 4, X repressor alone; 2, recApm; 3, recApm without ssDNA 5, recApm36; 6, recApm36 without ssDNA.

7

0 2 4 6 8 IO 12 PROTEIN (pmot X 10-l)

FIG. 6. Retention of single-strand DNA on filters. Reaction mixtures (50 pl) containing 20 mM Tris-HCI, pH 7.5, 12 mM MgC12, 10 p~ [3H]ssDNA were incubated for 10 min at 37 “C and placed on BA-85 nitrocellulose filters previously equilibrated in the Tris/Mg buffer. The filters were washed with 1 ml of buffer, dried, and counted in 5 ml of Betafluor (National Diagnostics). 0, recAec; 0, recAec1; ., recApm; 0, recApm36; X, recAec25.

was incubated with recAec protein, ATP, and magnesium, the DNA was aggregated to a form which could be sedimented from the solution. Duplex DNA could be incorporated into large aggregates by recAec but only in the presence of single-

Functional Organization of recA Protein 953

0 5 IO 15 5 IO I5

PROTEIN (pmol X 10-9

FIG. 7. Retention of duplex DNA on filters. 0, reaction mixtures were incubated and filtered as described in the legend to Fig. 7 except blunt end linear duplex DNA was substituted for ssDNA. 0, mixtures also contained 1 mM ATP and an ATP-regenerating system consisting of 8 units/ml phosphocreatine kinase and 2 mM phosphocreatine. A , recAecl; B, recAec; C, recApm36; D, recApm.

100, I I

" a z

recA PROTEIN ( f l )

n ac l o o -

80 -

60 - 40 - 20 -

I I I I I I I,,, I I I 0 I 2 0 IO 20 30 40 50 100 20r)

PROTEIN (pM) [KCI] (mM)

FIG. 8. Aggregation of DNA by recA proteins. Reaction mixtures (100 pl) containing 20 mM Tris-HC1, pH 7.5, 25 mM MgCl2, 1 mM ATP, and ATP regeneration as described in the legend to Fig. 7 were incubated for 10 min at 37 "C. Samples were centrifuged at 15,000 X g for 30 s and the radioactive DNA remaining in the supernatant was determined. A: 6.6 p~ ssDNA; 0, recAec; 0, recAec1. B: 6.6 p~ ssDNA and 5.7 p~ duplex [32P]DNA; 0, recAec; 0, recAecl; X, recAec with duplex DNA alone. C and D: 5.7 PM duplex [32P]DNA; 0, recApm; 0, recApm36.

strand DNA (Fig. 8B). The single strand and duplex DNA molecules used in this experiment shared no sequence homology. recAecl was incapable of forming aggregates with either single strand or duplex DNA.

recApm and recApm36 were found to aggregate duplex DNA without addition of single-strand fragments. When the amount of protein was varied, we found a nonlinear relationship with respect to amount of protein required for aggrega-

tion of the duplex DNA (Fig. 8C). Duplex DNA aggregation by recApm was sensitive to monovalent cations as inhibition occurred at about 10 mM KC1 (Fig. 80). In sharp contrast to the intact protein, KC1 concentrations of 100 mM were required to cause inhibition of duplex DNA aggregation by recApm36. This observation again shows that recApm36 binds more tightly to duplex DNA than recApm. At KC1 concentrations that inhibit aggregation of duplex DNA alone, the addition of single-strand DNA allowed recApm to catalyze the aggregation of the duplex DNA (data not shown).

Aggregation of Duplex DNA Stimulates Ligation-The assay for DNA aggregation described above requires large aggregates and we sought an assay that would detect smaller, less stable aggregates. Pheiffer and Zimmerman (31, 32) reported that molecular "crowding" of restriction fragments in solution stimulated the rate of DNA ligation by DNA ligases. We therefore examined whether recA protein brought DNA molecules together in a manner that enhanced ligation of DNA ends. Linear 6x174 duplex DNA was generated by the restriction enzyme AuaII. T4 DNA ligase was incubated for 10 min at 37 "C with duplex DNA in the presence or absence of recA proteins. Ligase alone catalyzed linkage of only a small portion of the DNA molecules into either circular monomers or linear dimers (Fig. 9, lane 8). Addition of recAec, recApm, or recApm36 enhanced the formation of dimer sized product. recAecl was unable to stimulate ligation by the T4 enzyme and when mixed with recAec ablated the stimulatory activity of the intact protein (Fig. 9, lanes 2 and 3). Stimulation of ligation was not specific to AuaII restriction fragments, which have 3 base single-strand ends that are complementary, since recAec also enhanced the ligation of blunt end DNA fragments (data to be presented elsewhere).

recA Protein-catalyzed Strand Transfer-recA protein can catalyze the transfer of a DNA strand from a linear duplex molecule to a homologous single-strand circle (9, 33). Strand transfer proceeds efficiently when ATP is maintained by regeneration from ADP and inorganic phosphate (34) and E. coli ssb is present in the reaction mixture (33, 35). recAec promotes strand transfer in the presence of ssb while recAec1 does not (Fig. 10, lane 2 uersus 3). When sufficient recAec protein to promote strand transfer was mixed with an equal amount of recAecl protein followed by incubation at 37 "C, no intermediate or product structures could be observed after

1 2 3 4 5 6 7 8 9 - Origin

- Trimer Linear - Dimer Linear - Monomer Circle

"""" - Monomer Linear

1

FIG. 9. Aggregation of linear duplex DNA by recA protein enhances ligation by T4 DNA ligase. Reaction mixtures (50 pl) containing 20 mM Tris-HC1, pH 7.5, 1 mM ATP, 15 mM MgC12, 0.1 mg/ml bovine serum albumin, 0.5 p M recA protein, and 6.6 p~ 4x174 DNA digested with AvaII were incubated 10 min at 37 "C. T4 ligase was then added and incubation continued for 20 min. EDTA and SDS were added to 50 mM and 1%, respectively, and the samples were electrophoresed through a 1% agarose gel. Gels were dried and exposed to XAR-5 film. Lane 1, recAec; 2, recAecl; 3, recAec + recAecl; 4, recApm; 5, recApm36; 6, recApm + recApm36; 7, ligase alone; 8, no ligase; 9, 6x174 form I and I1 markers.


FIG. 10. Protein-catalyzed DNA strand transfer. Reaction mixtures (30 pl) containing 20 mM Tris-HCI, pH 7.5, 15 mM MgClp, 1 mM ATP, 4 units/ml phosphocreatine kinase, 1 mM phosphocreatine, 4 pM 4x174 single-strand circular DNA, and 3.3 p~ $X174 linear duplex ["PIDNA were incubated for 50 min at 37 "C. SDS and EDTA were added to 1% and 50 mM, respectively. Samples were electrophoresed through a 1% agarose gel and the gel was dried and exposed to XAR film.

2 3 4 5 6 7 8 9 1 0 1 1

+ + + + - + - + + " - " -

gel electrophoresis. Thus recAecl prevents the wild-type protein from producing a stable synaptic structure between circular single strand and duplex DNA molecules.

Strand transfer by recApm was also stimulated by E. coli ssb (11) and complete transfer of the linear strand occurred when the ATP concentration was maintained by regeneration (Fig. 10, lane 6). Fig. 10 also shows that recApm did not produce intermediate or product unless ssb protein was present (lane 5 uersus 6 ) . However, recApm36 produced an intermediate in the absence of ssb (lane 7) which migrated differ- ently than the intermediate observed in recApm reactions. Addition of ssb neither changed the mobility of this reaction intermediate nor promoted the generation of a nicked circular product band, indicative of complete strand transfer. Recent studies with the protein fragment recApm36 (to be presented elsewhere) have shown that complete strand transfer rather than just synapsis (Fig. 10, lane 8) can occur if reaction mixtures include 0.2 M KC1. Under standard reaction conditions strand transfer was apparently initiated by recApm36 but stalled at some stage prior to completing the exchange. Moreover, aberrant strand transfer was the result observed when an equal amount of recApm36 and recApm were mixed before the reaction was started (Fig. 10, lane 9).

DISCUSSION

This paper describes the production, isolation, and prelim- inary characterization of altered recA polypeptides. We com-

TABLE I Summary of protein interaction with ATP and DNA

The symbols are: +, wild type; -, nonfunctional; A, altered; ds, double-stranded.

recAec recAl recA25 recApm recA36

Binds ATP + + - + + Hydrolyzes ATP + - - + + Cleaves repressor + - - + + Binds ssDNA + + - + + Binds dsDNA + - - + A Aggregates dsDNA + - - + A Strand transfer + - - + A

intermediates - -nicked circle

- duplex linear - -form I - ss linear

+ - + + + - ssb """ - recAec

- recAec 1 """

+ + " + - - recAprn " + + + - - recAprn 36

pared the intact recA proteins from E. coli and P. mirabilis with: the recAecl protein, a 26,000-dalton recAec protein expressed from a truncated gene, and an isolated proteolytic fragment of the recApm protein (recApm36). Results of experiments detailing protein interaction with ATP and DNA are summarized in Table I.

Partial proteolysis by the proteases subtilisin or V8 produced peptide fragments of similar size from both recAec and recApm proteins. Many tryptic fragments generated from recApm protein are identical in amino acid content to tryptic fragments produced from recAec (data not shown). This is consistent with the findings that recombinant plasmids containing the recA gene of P. mirabilis restore UV resistance to E. coli cells carrying the recAl allele (13) and that recApm protein could catalyze cleavage of E. coli lexA and X CI proteins in vitro (29).

The product of the recAI6 allele, recAec26 protein, which shows a dominant mutant phenotype in uivo (20) was purified and examined for the ability to bind ATP and DNA. The recAec26 peptide did not bind ATP or DNA and did not interfere with the binding of these cofactors by the intact protein. The lack of negative complementation in uitro may reflect differences between in uitro biochemical assays and conditions in uiuo. Alternatively, a property of recA protein critical to providing UV resistance may include some protein interaction that does not involve the ATP or DNA binding sites. We cannot at present distinguish between these possi- bilities.

The recAl allele has been extensively characterized both in vivo and in uitro. On a multicopy plasmid the recAl allele is dominant over the chromosomal recA gene. The gene product contains a single amino acid change at position 160 (28) that alters the ability of the protein to interact with ATP and DNA (30). Although previous studies of recAecl showed that the protein did not function as an ATPase (8) we found that the protein binds ATP in a specific manner. recAecl protein cannot aggregate duplex DNA and interferes with the ability of wild-type protein to aggregate DNA or catalyze strand transfer to form heteroduplex DNA. This provides a biochemical basis for the dominant recombination deficiency observed

Functional Organization of recA Protein 955

in vivo. Most striking is the observation that recAecl bound single-strand DNA with the same stoichiometry as wild-type protein but did not bind duplex DNA.

recA protein bound duplex DNA and retained the DNA on filters. This binding was observed after incubation at 37 "c with MgClz and a pH of 7.5. Another indication that recA protein binds duplex DNA in solution is that recA protein can enhance the rate of T4 ligase-catalyzed linkage of restriction fragments. Increasing the rate of ligation by DNA aggregation has previously been observed with synthetic polymers (32) and a DNA binding protein from Xenopus (36). recA protein can stimulate the ligation of staggered or blunt end fragments (data not shown), a property which may be of some use in gene cloning strategies.

Incubation of recAec, Mg, and ATP with single-strand DNA resulted in formation of large protein-DNA aggregates that can be sedimented from solution. recAec mediated aggregation of duplex DNA only occurred if single-strand DNA was also present, although the two DNA molecules need not be homologous. This apparent activation of duplex DNA binding and aggregation may be an important property for synapsis of recombining DNA molecules and may facilitate a rapid search for DNA homology.

A proteolytic fragment from recApm protein (recApm36) missing 8% of the amino acid residues from the carboxyl terminus was isolated and characterized. As summarized in Table I, recApm36 demonstrates duplex DNA binding properties that differ from those of the intact protein. While recApm36 can bind and hydrolyze ATP, cleave X repressor, and interact normally with single-strand DNA, we observed four distinct changes in the interaction of recApm36 with duplex DNA 1) retention of duplex DNA on filters was directly proportional to the amount of recApm36 protein even at low concentrations of protein, 2) duplex DNA was still retained on filters by recApm36 when ATP was present, 3) a 10-fold higher concentration of KC1 was required to inhibit recApm36 catalyzed aggregation of duplex DNA than was necessary to inhibit the intact protein, and 4) stable joint molecules between linear duplex DNA and single-strand DNA are formed by recApm36 in the absence of ssb. Functionally, recApm36 protein initiated but failed to complete the transfer of a DNA strand from linear duplex DNA to a single-strand DNA molecule (Fig. 10). Recent studies with the protein fragment recApm36 have shown that complete strand transfer rather than just synapsis (Fig. 10, lane 8) can occur if reaction mixtures include 0.2 M KCl. A complete analysis of strand transfer and other recombination reactions will be presented in a subsequent manuscript. Apparently recA protein can be altered in a manner that affects interaction with duplex DNA while single-strand DNA binding is normal.

A recent model describing a theoretical active site organization of recA protein suggests that a recA monomer has two DNA binding sites (2). The observations described in this paper could be explained within such a theoretical framework by assuming that single-strand DNA binding involves site I, double-strand DNA binding reflects site 11, and aggregation and strand transfer require both site I and 11. Thus we would predict that recAec1 has a nonfunctional site I1 and recApm36 is altered at site I1 in a manner that increases the stability of duplex DNA binding and prevents heteroduplex product formation. Additional experiments examining recombination structures formed by recApm36 may serve to delineate between the "two-site'' model mentioned above and other cur-

rent theories of the molecular mechanism of recA catalyzed recombination.

Acknowledgments-We would like to thank F. Sladek and Drs. J. Runnels and S. West for critical review of this manuscript.

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Isolation of Altered recA Polypeptides and Interaction with ATP and