Renaturation of DNA by a Saccharomyces cerevisiae protein that catalyzes homologous pairing and strand exchange.

A protein from mitotic Saccharomyces cerevisiae cells that catalyzes homologous pairing and strand exchange was analyzed for the ability to catalyze other related reactions. The protein was capable of renaturing complementary single-stranded DNA as evidenced by S1 nuclease assays and analysis of the reaction products by agarose gel electrophoresis and electron microscopy. Incubation of the yeast protein with complementary single-stranded DNA resulted in the rapid formation of large aggregates which did not enter agarose gels. These aggregates contained many branched structures consisting of both single-stranded and double-stranded DNA. These reactions required stoichiometric amounts of protein but showed no ATP requirement. The protein formed stable complexes with both single-stranded and double-stranded DNA, showing a higher affinity for single-stranded DNA. The binding to single-stranded DNA resulted in the formation of large protein:DNA aggregates. These aggregates were also formed in strand-exchange reactions and contained both substrate and product DNAs. These results demonstrate that the S. cerevisiae strand-exchange protein shares additional properties with the Escherichia coli recA protein which, by analogy, gives further indication that it might be implicated in homologous recombination.

The pairing of DNA molecules at homologous regions and subsequent exchange of DNA strands results in the formation of joint molecules containing regions of symmetric and/or asymmetric hybrid DNA. The strand-exchange reaction is probably the most extensively studied aspect of the enzymology of recombination. Most of our understanding comes from the pioneering work with the Escherichia coli recA protein, which is involved in recombination, DNA damage repair, and the regulation of the SOS response (for a review see Ref 10). The recA protein can catalyze the formation of hybrid DNA starting with a variety of different DNA substrates (as reviewed in Refs. 11,12). Mechanistically, one of the best understood processes is the formation of joint molecules between circular single-stranded DNA and linear doublestranded DNA (13-15, 19, 20). Initially, recA binds stoichiometrically to single-stranded DNA to form an active intermediate (16)(17)(18) in the formation of joint molecules. The development of the hybrid DNA ("branch migration") is polar and proceeds in a 3' to 5' direction with respect to the invading strand (19,20). The bacteriophage T4 uvsX protein has been shown to catalyze strand-exchange reactions in a similar fashion (21).
Proteins that catalyze strand-exchange have also been purified from eukaryotes (22)(23)(24)(25)(26)(27). The recl protein from Ustilago maydis catalyzes hybrid DNA formation in the 5' to 3' direction which is in the opposite direction to strand-exchange catalyzed by recA (28). An activity that catalyzes limited strand-exchange in the 5' to 3' direction has been purified from human cells (25). This activity does not require ATP, differing in this respect from the strand-exchange proteins described above.
Recently, our laboratory has purified and characterized a strand-exchange protein from mitotic S. cerevisiae cells (26). This protein shares a number of characteristics with the E. coli recA protein. The reaction requires homologous substrates and stoichiometric amounts of protein, and it shows a cooperative dependence on protein concentration. Joint molecules are formed by displacement of one strand of the linear duplex by the single-stranded circular molecule, and hybrid DNA formation occurs in the 3' to 5' direction, making the S. cerevisiae protein in this respect unique among the eukaryotic strand-exchange activities.
Whereas the involvement of the prokaryotic enzymes (recA, UVSX) in genetic recombination has been demonstrated by genetic evidence, the role of the eukaryotic strand-exchange activities in recombination can only be inferred by analogy to the recA or UVSX proteins. Although the recl protein is missing in U. m y d i s recl strains (29), a recombination deficient mutant, a firm gene-product relationship has not been established. In S. cerevisiae, a direct approach using reverse genetics to clone the gene encoding the strand-exchange protein and to study its in vivo function is now possible.
The renaturation of complementary single-stranded DNA fragments can be considered as the simplest pairing reaction. The E. coli recA protein was found to renature complementary DNA in an ATP-dependent fashion (30)(31)(32)(33). In this paper, we present evidence that the S. cerevisiae strand-exchange protein renatures complementary single-stranded DNA. The reaction was characterized by an S1 nuclease assay, and the products of the reaction were analyzed by agarose gel electrophoresis and electron microscopy. We also report data o n the DNA-binding properties of the S. cerevisiae protein and found that the protein aggregates DNA into large complexes. coli strain AB259 HfrH, thi-I, rel-I and bacteriophage M13mp19 were from laboratory stocks.

Strains
Enzymes and Chemicals-Restriction endonucleases were obtained from New England Biolabs (Beverly, MA) and used as suggested by the manufacturer. Creatine phosphokinase (Type I), creatine phosphate, calf thymus DNA (Type I) were from Sigma.
Nucleic Acids-To purify M13mp19 viral DNA, M13mp19 phage were obtained by standard methods and further purified by centrifugation in CsCl density gradients (26). M13mp19 viral DNA was extracted from the purified phage particles and M13mp19 RFI DNA was purified from infected cells as previously described (26). 3H-Labeled M13 DNA was obtained by growing the cells in 2 liters of Fraser's medium (34) supplemented with 0.01% thiamine to a density of O D~W = 0.15. Then 5 mCi of [3H]thymidine (20 Ci/mmol; Du Pont-New England Nuclear) and adenosine and deoxyadenosine, both to final concentration of 1 mM, were added. The cells were grown until the culture reached an ODs90 = 0.7, and then phage were added to a multiplicity of infection of 10. The preparation of single-stranded and double-stranded DNA was then carried out as described above. DNA concentrations are expressed as moles of nucleotides using an €260 of 6800 and 8500 for double-stranded and single-stranded DNA, respectively.
Purification of the Strand-exchange Activity-The strand-exchange activity was purified through Fraction V exactly as described in Kolodner et al. (26) and used throughout this work. The strandexchange protein was >90% pure, as judged by polyacrylamide gel electrophoresis, and had a protein concentration of 3 mg/ml or 13.4 mg/ml, depending on the preparation. We have also further purified this protein by additional chromatography on double-stranded DNA cellulose to achieve a purity of >95%.' Assay for DNA Renaturation-Assays were carried out in 15 pl as described for the strand exchange assay (26). The reaction contained 33 mM Tris-HC1, pH 7.5, 13 mM MgC12, 1.8 mM dithiothreitol, 1.3 mM ATP, 3 mM creatine phosphate, 88 pglml bovine serum albumin, 10 units/ml creatine kinase, and 0.15 nmol of Hind111 cleaved doublestranded M13mp19 [3H]DNA (37,000 cpm/nmol), which had been denatured by boiling for 3 min and quenching on ice. After the addition of enzyme, reactions were incubated for 5 min at 30 "C, unless otherwise stated.
For analysis of reaction products by agarose gel electrophoresis, 0.5 M EDTA, pH 8.0,20 mg/ml Proteinase K (Beckman Instruments; Palo Alto, CA) and 10% NaDodSO: were added to 50 mM, 600 pg/ ml and 0.1%, respectively, and the reactions were incubated at 37 "C for 10 min. Then ' / s volume of a solution containing 0.25% bromphenol blue, 0.25% xylene cyano1 FF, 120 mM EDTA, and 15% (w/ v) Ficoll was added, and each sample was analyzed by electrophoresis through an 0.8% agarose slab gel run in buffer containing 40 mM Tris acetate, pH 7.9, 1 mM EDTA, and ethidium bromide at 0.5 pglml.
For analysis of reaction products by digestion with S1 nuclease, the samples were diluted 33-fold with 500 pl of S1 buffer containing 50 mM sodium acetate, pH 4.7,300 mM NaC1, and 1 mM zinc acetate. Two-hundred units of S1 nuclease (Bethesda Research Laboratories) and 10 pg of heat-denatured calf thymus DNA were added, and the reactions were incubated for 30 min at 37 "C. In some experiments 5.4 pl of 10% NaDodS04 was added per 15 pl of renaturation/ protection reaction assay mix after the incubation period. Subsequent W.-D. Heyer and R. D. Kolodner, manuscript in preparation. The abbreviation used is: NaDodS04, sodium dodecyl sulfate. dilution into the S1 digestion assay yielded a final concentration.of 0.1% NaDodSOl during the S1 digestion. Then 20 pg of calf thymus DNA and 500 pl of ice-cold 10% (w/v) trichloroacetic acid were added, and the reactions were kept on ice for 5 min. The samples were filtered through GF/C filters (Whatman; Maidstone, England), and the filters were washed 5 times with 3 ml of ice-cold 1 M HCl, 0.1 M NaPPi and once with ice-cold ethanol. The filters were dried for 10 min under a infrared heating lamp, mixed with 5 ml of Betafluor (National Diagnostics; Somerville, NJ), and counted in a Beckman LS 7000 scintillation counter to determine the bound radioactivity.
Additionally, in some experiments the amount of S1-sensitive DNA was monitored as acid-soluble radioactivity. Instead of filtering the samples through GF/C filters, the samples were centrifuged for 10 min in an Eppendorf microcentrifuge at 4 "C, and 0.5 ml of the supernatant was mixed with 4 ml of Aquasol (Du Pont-New England Nuclear) and counted to determine the acid-soluble radioactivity. Under these conditions, the single-stranded DNA was degraded completely by S1 nuclease, leaving >95% of the radioactivity acid-soluble. Upon omission of S1 nuclease, >95% of the radioactivity was trapped on GF/C filters leaving <5% acid-soluble radioactivity. The S1 nuclease assay using complementary single-stranded substrate DNA is referred to as the DNA renaturation assay. The amount of renaturation is expressed as percent acid-precipitable radioactivity relative to a control where S1 nuclease was omitted. SI Protection Assay-As a control for the DNA renaturation assay using S1 nuclease, we used noncomplementary single-stranded, substrate DNA (M13mp19 viral [3H]DNA; 3900 cpm/nmol) under reaction conditions described above in an assay referred to as the S1 protection assay. In fact we found that at higher protein concentrations than required for DNA renaturation, a significant amount of noncomplementary single-stranded DNA was protected from degradation by S1 nuclease (see Fig. 2 A , m). The protection was sensitive to NaDodSO, treatment (see Fig. 2B) and was clearly different from DNA renaturation as defined by the formation of double-stranded, therefore S1 resistant, DNA, as discussed below. The amount of protection is expressed as percent acid-precipitable radioactivity relative to a control where S1 nuclease was omitted.
DNA Binding Assays-Fifteen pl of DNA renaturation reactions were carried out with either 0.15 nmol of single-stranded M13mp19 viral [3H]DNA (3,900 cpm/nmol) or 0.15 nmol of double-stranded M13mp19 RFI [3H]DNA (37,000 cpm/nmol) and incubated for 5 min at 30 "C. The samples were diluted 33-fold with 500 p1 of ice-cold wash buffer containing 33 mM Tris-HC1, pH 7.5, 13 mM MgC12, 1.8 mM dithiothreitol and filtered through KOH-treated nitrocellulose filters (BA85, 0.45 p~; Schleicher & Schuell) (35). The filters were washed with 1 ml of wash buffer and dried for 10 min under an infrared heating lamp, and then bound radioactivity was determined as described above.
Electron Microscopy-Ninety pl DNA renaturation reactions were carried through the Proteinase K digestion step as described above. Then the reactions were chromatographed on Pasteur pipet columns of Agarose A5m (Bio-Rad) run in 10 mM Tris-HC1, pH 8.0, 1 mM EDTA buffer containing 0.1 M NaC1. The DNA containing fractions were mounted for electron microscopy by the formamide technique essentially as described (36). Alternatively, 90 pl DNA renaturation reactions were carried through the Proteinase K step, and then the samples were electrophoresed through a 0.8% agarose gel. The renaturation product band and the corresponding region in the control lane (no enzyme added) were eluted by the "freeze-squeeze" technique (37), and the DNA was mounted for electron microscopy.
DNA Aggregation-Aggregation of DNA by the yeast strand-exchange protein was assayed as described (38,39). Briefly, 30-p1 aliquots were centrifuged for 4 min in an Eppendorf microcentrifuge. Three sequential aliquots of 9 pl were removed from the supernatant. The remaining 3 p1 and the pellet were suspended in 200 p1 of water. The amount of DNA in the total supernatant (30 pl) was estimated from the average radioactivity of the first 2 aliquots of the supernatant. The amount of radioactivity in the pellet was corrected for the presence of 3 pl of supernatant. to a high molecular weight form that remained at the top of an agarose gel (Fig. lA, lane 9) The product was not formed in the absence of protein (Fig. lA, lane 3). The generation of this product was fully dependent on the presence of MgClz (Fig. lA, lane 4) but did not require dithiothreitol (lane 5 ) or ATP (lane 7). The reaction was completely inhibited by 200 mM NaCl (Fig. L4, lane 8 ) , but the addition of 5 mM Nethylmaleimide had little or no effect (Fig. lA, lane 6). In these experiments the substrate DNA (linear, single-stranded DNA) was slightly fragmented due to the heat denaturation step and was not easily seen because it was a diffuse band that did not stain intensely with ethidium bromide. The reaction requirements were identical when lower amounts of protein (1 M , 132,000 polypeptide/264 nucleotides of singlestranded DNA) were used which resulted in only partial renaturation of the DNA as judged from the S1 assay (data not shown). The formation of this product was dependent on complementary DNA. When noncomplementary singlestranded M13mp19 viral DNA was used as substrate (Fig. 1B,  lanes 4-7) instead of complementary single-stranded DNA (Fig. lA), no high molecular weight product DNA was formed. This control also excludes the possibility that the product that remained at the top of the gel represented a protein:DNA complex that somehow withstood the NaDodSOJEDTA/Proteinase K treatment. Even a t higher protein concentrations (Fig. lB, lane 7), no ethidium bromide stainable material was detected at the top of the gel in experiments with noncomplementary single-stranded DNA. Since the reaction product of the renaturation reaction did not enter the gel, quantitation of the reaction by densitometry of the gel was not possible. Therefore, we attempted a quantitative approach of the reaction using an S1 nuclease assay.

The
Characterization of the DNA Renaturation Reaction-Since double-stranded DNA is resistant to S1 nuclease, it is possible to accurately quantitate the amount of renaturation by measuring the extent to which complementary single-stranded DNA becomes S1 resistant. A protein titration curve is illustrated in Fig. 2 (X). We found that complementary singlestranded DNA was converted to an S1 resistant form but that the interpretation of the data was complicated by the fact that a portion of the S1-resistant DNA could be explained by the protection of single-stranded DNA from degradation by S1 nuclease ( Fig. 2A and discussed in detail below). Therefore in Fig. 2A the curve labeled renaturation is in fact the sum of renaturation and protection explaining why 100% of the DNA could be rendered S1 resistant. We developed conditions under which addition of Na-DodSOI prior to digestion with S1 nuclease prevented the protection of single-stranded DNA (Fig. 2B). All of the data using the S1 digestion assay to quantitate renaturation that is discussed below (Fig. 2B, Fig. 3, and Table I) were obtained with the assay utilizing NaDodS04. The reaction showed that maximal renaturation occurred at 200 ng of protein/0.15 nmol of single-stranded DNA, or about 1 M , 132,000 polypeptide/ 100 nucleotides (assuming that all of the protein molecules were active). No more than 70% renaturation was observed in any experiment (Fig. 2B, Fig. 3) which is consistent with the structure of the renaturation products observed by electron microscopy (see below). Higher protein concentrations were not inhibitory, which is in contrast to the strand-exchange reaction (26). The renaturation reaction was fast; within 2 min of incubation a t 30 "C, most of the product was already formed (Fig. 3). The data also show that the protein did not act catalytically, since the products of the reaction reached limits proportional to the initial concentration of strand-exchange protein with identical kinetics. Thus at half the protein concentration, the maximum of renaturation was half; at 100 ng maximally 48% of the DNA was S1 resistant, at 50 ng 19%, and at 25 ng 8%. The requirement for different components in the reaction was determined using the S1 nuclease assay (Table I). DNA renaturation required M F .
The reaction was inhibited by 200 mM NaCl, but not by 5 mM N-ethylmaleimide. There was also no requirement for ATP or an ATP regeneration system. Results obtained with the S1 nuclease assay and the agarose gel electrophoresis  0 0.1 0.2 0 3 0.4 0.8 0.0 0.1 0.2 0 3 0.4 0 8

Protein Ugl
FIG. 2. Protein titration of DNA renaturation versus S1 protection. Reactions did not contain ATP and the ATP regeneration system and were incubated for 2 min a t 30 'C. All reactions were identical except that the complementary single-stranded DNA in the DNA renaturation assay was replaced by noncomplementary singlestranded DNA in the S1 protection assay. DNA renaturation assay ( x ) , S1 protection assay (W). A, the digestion with SI nuclease was performed in the absence of NaDodSO,. B, the digestion with S1 nuclease was performed in the presence of NaDodSO, as described under "Materials and Methods."

TABLE I Reaction requirements of DNA renaturation
DNA renaturation was quantitated using the S1 nuclease assay in the presence of NaDodSOc as described under "Materials and Methods." Reaction mixtures were incubated for 5 min a t 30 "C and contained 75 ng of Fraction V. The relative activity value of 100% is equivalent to 59% renaturation. assay were completely consistent with one another. Structure of DNA Renaturation Products-The DNA renaturation products were examined by electron microscopy. Complementary single-stranded DNA that had been incubated with the strand-exchange protein was found predominantly (>95% of the mass) in the form of complex aggregates (Fig. 4, A and B ) . The aggregates consisted of double-stranded and single-stranded stretches of DNA and were resistant to the NaDodS04/EDTA/Proteinase K treatment. No differences were found when the reaction products were purified from an agarose gel and then spread for electron microscopy or when the entire reaction was spread directly after the NaDodS04/EDTA/Proteinase K treatment and filtration through Agarose A5m. Reaction mixtures that were incubated without protein did not show these large aggregates (see Fig.  1 ), and when spread directly for electron microscopy, only unreacted substrate DNA (i.e. linear single-stranded DNA) could be detected in the electron microscope (Fig. 4C). These results indicate that the yeast strand-exchange protein catalyzed the renaturation of single-stranded DNA, resulting in the rapid formation of large aggregates that consisted mostly of duplex DNA.

Relative activity
Protection of Noncomplementary Single-stranded DNA from SI Nuclease Digestion-In control experiments for the S1 nuclease assay, we replaced the complementary singlestranded DNA (heat-denatured, linear, double-stranded DNA) of the DNA renaturation assay with noncomplementary single-stranded DNA (M13mp19 viral DNA) in the S1 protection assay. It was noted that at high strand-exchange protein concentrations significant protection of the noncomplementary single-stranded M13mp19 viral DNA from S1 nuclease digestion occurred, despite the fact that no renaturation of such DNA was seen by agarose gel electrophoresis (see Fig. 1). The result of a protein titration using the S1 protection assay is shown in Fig. 2A (M). The protection reaction required significantly greater amounts of strandexchange protein than the renaturation reaction. The reaction requirements for this reaction were found to be similar to the requirements for the renaturation reaction (see Table I) except that the S1 protection was largely independent of a requirement for Mg+. The protection reaction was clearly different from the renaturation reaction, since the effect was completely eliminated if NaDodS04 was added prior to the S1 digestion to disrupt protein:DNA complexes (Fig. 2 B ) . Also, the time courses of both reactions at a given, limiting protein concentration were found to be different. The renaturation reaction was fast and almost complete at 2 min (see Fig. 3), whereas the protection reaction reached a maximum only after 40 min (data not shown). As discussed below we believe that the extended time course for the protection reaction is due to the packaging of the DNA into a large protein:DNA complex rather than being mediated by the simple binding of the protein to the DNA. DNA Binding Properties of the S. cerevisiae Strand-Exchange Protein-The protein formed stable complexes with both single-stranded and double-stranded DNA. Fig. 5 shows the result of a protein titration experiment measuring the binding of stable protein:DNA complexes to nitrocellulose filters. Maximal complex formation between Fraction V and single-stranded DNA was observed at about 100 ng of protein/ 0.15 nmol of single-stranded M13mp19 viral DNA. The affinity of Fraction V for double-stranded DNA was much lower than for single-stranded DNA (Fig. 5). Maximal complex formation occurred at about 375 ng of Fraction V/0.15 nmol of double-stranded DNA. No further increase in complex formation was measured using up to 750 ng of protein (data not shown). The reaction requirements for protein:DNA complex formation were determined, and the results are presented in Table 11. The formation of stable protein:DNA complexes did not require Mg2' and was only partially inhibited by Nethylmaleimide. There was also no requirement for ATP or an ATP regeneration system. The salt stability of the pro-

TABLE I1
Reaction requirements of DNA:protein complex formtion DNAprotein complex formation was measured as described under "Materials and Methods." Reaction mixtures (15 pl) were incubated 5 min at 30" C and contained 75 ng of Fraction V in reactions with single-stranded DNA and 375 ng of Fraction V in reactions with double-stranded DNA. The relative activity value of 100% is equivalent to 83% of the radioactivity bound with single-stranded DNA and 85% of the radioactivity bound with double-stranded DNA.  60 pl were removed. The first aliquot was subjected to the S1 protection assay, determining the amount of DNA that was sensitive to digestion by S1 nuclease in the absence of NaDodS04. Control reaction without protein, ( x ) ; reaction with protein, (W). The second aliquot was centrifuged for 4 min and the radioactivity in the supernatant and in the pellet was determined as described ( (26). Lane 3, reaction with protein; lane 4, reaction without protein. Additional aliquots were assayed for DNA aggregation and S1 nuclease sensitivity as described in the text. tein:DNA complexes was assessed by measuring the amount of complex bound to filters with various concentrations of NaCl present in the incubation mix (data not shown). The complexes between single-stranded DNA and Fraction V were more stable than the double-stranded DNA:Fraction V complexes. At 100 mM NaCl, for example, 78% of the singlestranded DNA:protein complexes persist but only 52% of the complexes with double-stranded DNA. This difference was also reflected in the elution profiles of the 132,000 k polypeptide from single-stranded DNA and double-stranded DNA cellulose columns,3 where the strand-exchange protein elutes a t about 250 mM NaCl and at about 100 mM NaCl, respectively.
Aggregation of DNA by the Yeast Strand-exchange Protein-As discussed below, we reasoned that the protection of noncomplementary, single-stranded DNA from S1 nuclease digestion by the yeast strand-exchange protein was not mediated by simple covering of the DNA by the protein as in a footprint type of protection. Therefore, we tested whether the protein sequesters DNA in large aggregates rendering the DNA S1 resistant. Incubation of the yeast strand-exchange protein with noncomplementary single-stranded DNA resulted in the formation of large protein:DNA aggregates, which sedimented a t greater than 10,000 S (38). The formation of these aggregates had the same kinetics as the protection of the DNA from S1 nuclease digestion (Fig. 6A)  ured as a decrease in the amount of S1-sensitive DNA present. The addition of NaDodSOI to 0.1% completely disrupted these aggregates (data not shown). This suggests that it is the formation of these large aggregates which renders the DNA inaccessible to S1 nuclease.
In a strand-exchange reaction, the protein coaggregated homologous, linear double-stranded and circular singlestranded DNA, as illustrated in Fig. 6B. The DNA in the reaction was quantitatively aggregated and present in the pellet (compare lune 1 with lune 3). The small amount of substrate DNA (linear double-stranded and circular singlestranded DNA) in the control reaction (lane 2 ) was due to the presence of the residual 3 pl of supernatant in the pellet fraction. Assays of parallel aliquots showed that the 3Hlabeled single-stranded DNA was protected from S1 nuclease digestion. Furthermore, the aggregation assay confirmed that all radioactivity was present in the pellet leaving no radioactivity in the supernatant. Interestingly, as seen in Fig. 6B, lane I, the aggregates contain substrate DNA (linear doublestranded and circular single-stranded DNA) as well as product DNA (joint molecules).

DISCUSSION
We have shown that the S. cerevisiue strand-exchange activity renatures complementary single-stranded DNA. Three independent pieces of evidence support this notion: 1) the yeast activity forms a new DNA species on agarose gels, after incubation with complementary single-stranded DNA, which is reminiscent of the renaturation products formed by the E. coli recA protein (30); 2) the yeast activity transforms complementary single-stranded DNA into an S1 nucleaseresistant form under conditions where protection of noncomplementary DNA was eliminated by treatment with Na-DodS04, which has been a classic definition of DNA renaturation (30)(31)(32)(33); and 3) renaturation products, when examined by electron microscopy, were shown to be large aggregates containing double-stranded DNA which were very similar to the products formed by recA (30).
The DNA renaturation reaction can be regarded as the simplest model for the formation of hybrid DNA and will be compared in the following to the strand-exchange reaction performed by the same activity. All the assays described in this paper were performed under strand-exchange conditions (as used in Ref. 26) to simplify this comparison. Both reactions required stoichiometric amounts of proteins, a different stoichiometry is required, however. Maximum strand-exchange required 1 monomer/l2-14 nucleotides of singlestranded DNA (26), whereas maximum DNA renaturation was achieved by 1 monomer/100 nucleotides. This is more protein than expected from a mechanism in which the yeast protein simply provides a nucleation point for DNA renaturation. The reaction requirements are identical with the notable exception that N-ethylmaleimide inhibits strand-exchange completely but has no effect on DNA renaturation. This suggests that the molecular mechanisms for both reactions are not identical and that there is a different requirement for sulfhydryl-group(s).
The yeast strand-exchange activity forms stable complexes with single-stranded and double-stranded DNA. It has a high affinity for single-stranded DNA, whereas the affinity to double-stranded DNA is much lower. It is not clear at this point whether the yeast strand-exchange protein binds first to single-stranded DNA in the strand-exchange reaction as was shown for the recA protein (16)(17)(18). The stability of the protein:single-stranded DNA complexes at 200 mM NaCl (more than 70% of the complexes persist) suggests that the sensitivity of the DNA renaturation and strand-exchange reaction to NaCl at this concentration is not due to the simple disruption of the protein:single-stranded DNA complexes.
The yeast strand-exchange activity was found to protect single-stranded DNA from digestion by S1 nuclease at high protein concentrations. Since the S1 nuclease assay is widely used to demonstrate and quantitate DNA renaturation, it should be pointed out that this protection from S1 nuclease digestion might be a source of artifacts in DNA renaturation assays where a control with noncomplementary singlestranded DNA has not been performed or where the protein has not been dissociated from the DNA prior to S1 digestion. The significant protection of single-stranded DNA at a stoichiometry of 1 M, 132,000 polypeptide/100 nucleotides suggests that there is not enough protein in the reaction to completely cover the DNA for a footprint-type of protection. The strand-exchange reaction, for comparison, requires 1 M , 132,000 polypeptide/l2-14 nucleotides. In a time course of S1 protection at even lower protein concentrations (1 M, 132,000 polypeptide/440 nucleotides) the protection effect was low at 5 min (19%) but steadily climbed to a maximum of 78% at 40 min. This slow time course also suggests that the S1 protection reaction is not mediated solely by simple binding of the protein to DNA because binding to DNA as measured in the nitrocellulose filter binding assay is completed within 2 min (data not shown). This interpretation is strengthened by the observation that the activity forms fast sedimenting aggregates with single-stranded DNA at high protein concentrations which could render the single-stranded DNA inaccessible to S1 nuclease. This is also suggested by the parallel time course of the DNA aggregation and S1 protection reactions. Additionally, these aggregates were also found to be sensitive to detergent as was the S1 protection. This aggregate formation i s reminiscent of the network formation of DNA and recA protein that was discovered by Radding and co-workers (38,39). The yeast strand-exchange activity will aggregate DNA under strand-exchange conditions. Since we find that the DNA is quantitatively aggregated, we hypothesize that this is an early step in the strand-exchange reaction. It is clear that these aggregates are actively undergoing strandexchange since their formation is complete within 5 min, whereas strand-exchange proceeds in a linear fashion for 60 min (26). This view is supported by the observation that product DNA (joint molecules) is present in the aggregates. Reconstitution experiments with active intermediates will be needed to prove whether the aggregates are obligatory intermediates for the strand exchange reaction.
The additional properties of the S. cerevisiue strand-exchange activity reported here are similar to properties of previously characterized strand-exchange proteins, most notably to the E. coli recA protein. Thus far the only significant difference between the yeast protein and recA is the lack of an ATP requirement by the yeast activity for any of the reactions studied. A possible explanation for this is discussed in Kolodner et ul. (26). Due to the lack of genetic evidence we have to rely on these analogies to the recA protein as an initial indication that this S. cerevisiue activity is involved in homologous recombination. Recently, we have cloned a gene that potentially encodes the strand-exchange protein4 and are using the reverse genetic approach available in this organism to establish a gene-protein-function relationship.