Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes. To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity. The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA. In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed. Similar to the E. coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E. coli single-stranded DNA-binding protein. In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA.

The SGS1 gene of Saccharomyces cerevisiae was identified in a search for extragenic suppressors of the slow growth phenotype of cells deficient in DNA topoisomerase III, the product of the TOP3 gene (1). Paradoxically, whereas null mutations in SGS1 suppress the growth defect of top3 mutants, they significantly reduce the growth rate of top1 mutants lacking a functional DNA topoisomerase I (1)(2)(3). Based on the results of two-hybrid screens in yeast (4), it was suggested that Sgs1 protein interacted directly with DNA topoisomerase III (1) and perhaps DNA topoisomerase II as well (5). Yeast sgs1 mutants show increased genome instability; the rate of mitotic recombination between homologous sequences is elevated (1,5,6), and chromosome missegregation occurs more frequently during mitosis and meiosis (5,6). These characteristics are reminiscent of those of yeast top3 mutants lacking DNA topoisomerase III; in addition to a slow growth phenotype, top3 mutants show an elevated frequency of recombination between repetitive sequences and are defective in sporulation (1,7). The hyper-rec phenotype of top3 mutants is also suppressed by mutations in SGS1 (1).
The sequence of the Sgs1 protein suggests that it possesses a central region homologous to the Escherichia coli RecQ helicase (8,9), and a helicase activity was detected in a rabbit reticulocyte coupled transcription/translation system expressing the Sgs1 protein (2). The plausible association between the yeast Sgs1 helicase and DNA topoisomerase III, a member of the type IA subfamily of DNA topoisomerases that also includes E. coli DNA topoisomerases I and III, is reminiscent of an enzyme termed "reverse gyrase," which was previously found only in thermophilic organisms (for a review, see Ref. 10). Reverse gyrase catalyzes positive supercoiling of DNA, and its sequence suggests that it possesses both a DNA helicase and a type IA DNA topoisomerase (Refs. 11-13; for the classification of DNA topoisomerases, see Ref. 14). In the case of the Sulfolobus acidocaldarius enzyme, the putative helicase and the topoisomerase activity are present on the same polypeptide (11).
The association between these activities in a single enzyme led to the proposal that the enzyme acts by using its helicase activity to unwind DNA, generating both positive and negative supercoils; removal of the negative supercoils by the topoisomerase activity then results in a net accumulation of positive supercoils (11).
Recently, several additional homologues of the E. coli RecQ and yeast Sgs1 protein have been discovered. These include the products of the human Bloom's syndrome and Werner's syndrome genes (BLM and WRN, respectively) (15,16). Although the clinical features of the two diseases are rather different, patients of both syndromes exhibit chromosome instability and a predisposition to cancer (for reviews, see Refs. 17 and 18). BLM and WRN proteins are similar to Sgs1 protein in size and share limited sequence homology outside of the central helicase domain, suggesting that these RecQ-type proteins might be functionally related. A Schizosaccharomyces pombe homologue of SGS1, termed rqh1 ϩ , was also reported recently (19). Similar to SGS1 of the budding yeast, rqh1 ϩ has been shown to suppress recombination, especially during S phase arrest (19). Interestingly, the E. coli RecQ protein has also been shown to suppress illegitimate recombination (20).
As a first step in studying the mechanistic and functional aspects of the Sgs1 protein, we have purified a truncated form of it containing the helicase domain. We report here the DNAdependent ATPase and helicase activities of the purified protein.

Plasmids for Overexpression of Sgs1 Protein and Its Truncations-
The regions of the SGS1 gene encoding the N-and C-terminal segments of the protein were amplified by the polymerase chain reaction (PCR) 1 from pRS414-SGS1 (a gift from Dr. R. Sternglanz, State University of New York at Stony Brook). The N-terminal coding segment was amplified using the VENT polymerase (New England Biolabs) and a pair of * This work was supported by National Institutes of Health Grant GM24544 and fellowships from the Damon Runyon-Walter Winchell Foundation and the Human Frontier Science Program (to R. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  oligodeoxynucleotides RB1 (5Ј-GAGTCTACGGGATCCGTAACCATGG-TGACGAAGCCGTC-3Ј) and RB4 (5Ј-GGGGATCAAGCTTGCGGCAGT-GTACGAGAGC-3Ј). The sequence of RB1 was designed to place a BamHI site GGATCC (underlined) six base pairs upstream of the ATG initiation codon of SGS1, so that the final construct could be moved into a plasmid previously constructed for the overexpression of several proteins in yeast. The sequence of RB4 was designed to add a HindIII site AAGCTT (underlined) to the end of the N-terminal segment for the convenience of cloning. The C-terminal coding segment of SGS1 was similarly amplified using the primers RB2 and RB5 (5Ј-GGGGCATAA-GCTTCGGCCACGATAGCAGATCT-3Ј and 5Ј-GCTGGACTAGCTCGA-GCCTTTCTTCCTCTGTAGTGACC-3Ј, respectively). RB5 was designed to link the C-terminal codons of SGS1 to the sequence 5Ј-CTCGAGG-GGTA(CAC) 6 TAG-3Ј in an expression vector pG1TT, via the XhoI site CTCGAG (underlined in both sequences), so that a stretch of six histidines would be added to the C terminus of Sgs1 protein. The expression vector pG1TT 2 is a derivative of YEpTOP2-PGAL1, a plasmid originally constructed for the overexpression of yeast DNA topoisomerase II from the inducible GAL1 gene promoter (21). The N-terminal PCR product was cloned between the BamHI and HindIII restriction sites of pBluescript (Stratagene) to give pRB104. The C-terminal PCR product was then introduced between the HindIII and XhoI restriction sites of pRB104 to give pRB111. The segment in pRB111 between the AflII site in the N-terminal coding region of SGS1 and the NarI site in the C-terminal coding region of SGS1 was then replaced by the AflII to NarI fragment from pRS414-SGS1. DNA sequencing confirmed that the resulting plasmid, pRB121, contained the correct wild-type SGS1 coding region. The BamHI to XhoI segment of pRB121 was placed between the same sites in pG1TT for the expression of full-length Sgs1 protein with a hexahistidine tag at its C terminus.
Several constructs expressing truncated SGS1 fragments were derived from pRB121 and tested for overexpression of the encoded proteins. The one used in the work reported here, which expresses a recombinant Sgs1 protein comprised of amino acids 400 -1268 of the protein, was produced from pRB121 by PCR through the use of the primer pair RB8 (5Ј-GAGTCTACGGCGCGCCCGGATGTCGAAAGAG-3Ј) and RB11 (5Ј-GCTGGACTACTCGAGCCGAGCGTAAATCGCT-3Ј). In the PCR product, codons 400 -1268 of yeast SGS1 were placed between a BssHII and a XhoI restriction site (underlined) in the primer sequences. The BssHII to XhoI segment was then inserted into pG1TT. The final product, pRB222, is expected to express a recombinant protein MSTDPVYPYDVP-Sgs1-(400 -1268)-RRAVH 6 . Among the extra 12 amino acids at its N terminus, the first six are derived from the N terminus of yeast DNA topoisomerase II and the remaining six comprise the flu virus hemagglutinin epitope (HA), which was introduced into the recombinant protein to facilitate its detection by immunoblotting with a commercially available monoclonal antibody. The hexahistidine tag at the C terminus was added to facilitate the purification of the recombinant protein.
Purification of the Recombinant Sgs1 Fragment-pRB222 was first transformed into a protease-deficient yeast strain BCY123 (Ref. 22; strain was originally obtained from Dr. R. Kornberg's laboratory, Stanford University). A colony was picked and grown overnight in minimal medium lacking uracil and supplemented with 2% (w/v) glucose. This culture was used to inoculate 100 ml of uracil-minus minimal medium lacking glucose. After reaching saturation, the culture was diluted 100-fold into yeast YEP medium supplemented with 2% (w/v) lactic acid and 3% (v/v) glycerol and placed on a platform orbital shaker (New Brunswick) in a 30°C chamber. When a cell density of approximately 5 ϫ 10 6 cells/ml was reached, the GAL1 promoter was induced by the addition of galactose to 2% (w/v). Expression of the recombinant protein was allowed to continue for a further 8 h at 30°C. Cells were harvested by centrifugation for 10 min at 4°C and 5000 revolutions/min, washed, and then resuspended in an equal volume of buffer A (20 mM Tris⅐HCl, pH 8.0, 10% glycerol) plus 150 mM NaCl and a mixture of protease inhibitors (phenylmethylsulfonyl fluoride, 1 mM; leupeptin and pepstatin, 2 g/ml each; benzamidine, 20 g/ml). Triton X-100 was added to 0.1%, and the cells were lysed by the addition of an equal volume of acid-washed glass beads (Sigma 425-600 m) and blending in a Bead-beater (Biospec Products). In some preparations, lysis was done by vortexing the mixture vigorously (21). The cell extract was separated from the glass beads and other sediments by decantation, and the beads were washed twice in buffer A. The cell extract and washes were combined and clarified by centrifugation.
The crude lysate was loaded directly onto a 60-ml DEAE column equilibrated in buffer A. After washing the column with the same buffer, bound proteins were eluted in buffer A plus 300 mM NaCl. The eluate was then applied directly to a 30-ml heparin column equilibrated with buffer A plus 300 mM NaCl. Bound proteins were eluted with a 300-ml linear gradient of NaCl (0.3-1 M) in buffer A or, in some preparations, by a 1 M NaCl step elution. Fractions containing the truncated Sgs1 protein were identified by SDS-polyacrylamide gel electrophoresis. Pooled fractions were diluted with buffer A to a concentration equal to buffer A plus 0.5 M NaCl, and imidazole was added to 5 mM. The partially purified protein solution was then mixed with nickel-agarose beads (His-Bind resin, Novagen) to allow the binding of the hexahistidine-tagged protein to the beads. The beads were then washed several times in buffer A containing 0.5 M NaCl and 5 mM imidazole. After a final wash step with buffer A plus 0.5 M NaCl and 23 mM imidazole, the recombinant Sgs1 fragment was eluted in buffer A containing 100 mM NaCl and 200 mM imidazole. The eluted protein was dialyzed against a storage buffer (20 mM Tris⅐HCl, pH 8.0, 50 mM NaCl, 40% glycerol, 1 mM dithiothreitol). The final yield of the protein, estimated by the use of a protein assay kit (Bio-Rad) with bovine serum albumin solutions as standards, was between 1 and 2.5 mg for several preparations. Nucleic Acid Substrates-DNA oligomers (GENEMED) were purified by polyacrylamide gel electrophoresis when necessary. The RNA oligomer was purchased from Dalton Chemical Laboratories. Synthetic DNA substrates were prepared from three oligonucleotides 5Ј-GCCGT-GATCACCAATGCAGATTGACGAACCTTTGCCCACGT-3Ј (oligonucleotide 1), 5Ј-GACGTGGGCAAAGGTTCGTCAATGGACTGACAGCTG-CATGG-3Ј (oligonucleotide 2), and 5Ј-GCCATGCAGCTGTCAGTCCAT-TGACGAACCTTTGCCCACGT-3Ј (oligonucleotide 3). Oligonucleotides 2 and 3 were annealed to form the 40-base pair double-stranded DNA substrate with a one nucleotide overhang at the 5Ј-ends, and oligonucleotides 1 and 2 were annealed to form the forked DNA substrate with a double-stranded segment with single-stranded tails at one end (complementary regions in oligonucleotides 1 and 2 are underlined). In each case, oligonucleotide 2 was 32 P-labeled at its 5Ј-end, using T4 polynucleotide kinase and [␥-32 P]ATP. 32 P-Labeled oligonucleotide 2 was also used alone as the single-stranded DNA substrate. DNA substrates for testing helicase activity were constructed by the annealing of a complementary DNA strand with single-stranded X174 virion DNA. Short strands 26, 52, and 66 nucleotides in length were obtained by synthesis and were labeled at their 5Ј-ends by treatment with T4 polynucleotide kinase and [␥-32 P]ATP or at the 3Ј-ends by treatment with terminal transferase and ␣-32 P-labeled 3Ј-dATP. The 52-and 66-mers were complementary to X174 at nucleotides 130 -181 and 5357-36, respectively, and the sequence of the 26-mer DNA was the same as oligonucleotide 9 in Whitby et al. (24). Longer strands were derived from the 140-base pair AvaII-DraIII and the 558-base pair StuI-AvaII restriction fragments of the double-stranded replicative form of X174. These fragments were 32 P-labeled at their 5Ј-ends and purified on a nondenaturing 6% polyacrylamide gel. The electroeluted fragments were then denatured and annealed with X174 virion DNA. Conditions for DNA annealing and purification of the products were described previously (23). Single-stranded X174 virion DNA and supercoiled X174 replicative form DNA were purchased from New England Biolabs. Relaxation of supercoiled X174 DNA by vaccinia virus topoisomerase was carried out at 37°C in 40 mM Tris⅐HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA. The relaxed DNA was phenol-extracted and ethanol-precipitated before use.
ATPase Assay-Reactions (20 l each) were carried out in ATPase buffer (20 mM Tris⅐HCl, pH 7.5, 2 mM MgCl 2 , 0.1 mM ATP, 2 mM dithiothreitol, and 100 g/ml of bovine serum albumin) for 30 min at 30°C. Each reaction contained 25 nCi of [␥-32 P]ATP and the amounts of recombinant Sgs1 protein and DNA specified under "Results." Reactions were stopped by the addition of 1 l of 0.5 M EDTA to each, and 1 l of each reaction was spotted onto CEL 300 PEI/UV 254 thin layer chromatography plates (Sigma). The plates were developed in 1 M formic acid and 0.5 M LiCl, and the amount of ATP hydrolyzed in the reaction mixture was evaluated from the dried plates using a phosphor imager (Fuji).
Helicase Assay-Reactions (20 l each) contained annealed substrate DNA and were carried out as for the ATPase assay, except 2 mM unlabeled ATP was used. Reactions were stopped by the addition of 5 l of a stop buffer (0.5% proteinase K, 100 mM Tris⅐HCl, pH 7.5, 200 mM EDTA, 2.5% SDS) followed by incubation at 37°C for 10 min. Products were analyzed either by electrophoresis in 1% agarose (in a buffer containing 40 mM Tris acetate, pH 8.0, 1 mM EDTA) or polyacrylamide (in 89 mM Tris borate, pH 8.3, 2 mM EDTA). Agarose gels were dried on Whatman DE81 paper, and polyacrylamide gels were dried on Whatman 3MM paper. The dried gels were analyzed using a phosphor imager 2 R. Hanai and J. C. Wang, unpublished results.
Sgs1 DNA Helicase Activity of S. cerevisiae (Fuji). DNA concentrations refer to that of the X174 virion DNA in nucleotides. The E. coli single-stranded DNA binding protein (Ssb) used in some of the helicase assays was purchased from Promega.
DNA Binding Assays-To examine the binding of the recombinant Sgs1 fragment to either single-stranded or supercoiled replicative form X174 DNA, the indicated amounts of protein and DNA were incubated in 20 l of DNA binding buffer (20 mM Tris⅐HCl, pH 7.5, 2 mM dithiothreitol, 2 mM MgCl 2 , and 100 g/ml bovine serum albumin). Reaction mixtures were incubated at 20°C for 15 min, and the DNA-protein complexes were analyzed by electrophoresis in 1% agarose in 89 mM Tris borate, pH 8.3, 2 mM EDTA. DNA and DNA-protein complexes were visualized by staining with 1 g/ml ethidium bromide.
To compare the binding of Sgs1 with single-stranded, doublestranded, or forked DNA, binding reactions (20 l) containing 32 Plabeled DNA (0.1 M) and recombinant Sgs1 were incubated at 20°C for 15 min in the DNA binding buffer. Products were assayed by electrophoresis in a 6% nondenaturing polyacrylamide gel in 89 mM Tris borate, pH 8.3, 2 mM EDTA. Gels were dried, and the DNA was visualized using a phosphor imager (Fuji).

Purification of a Recombinant Sgs1
Protein-Initial attempts to purify full-length Sgs1 protein were unsuccessful because of the insolubility of the protein when overexpressed in S. cerevisiae. To circumvent this problem, three plasmids were constructed for the overexpression of truncated proteins from an inducible yeast GAL1 gene promoter. One plasmid was designed for expressing amino acid residues 1-1119, a second for amino acids 400 -1268, and a third for amino acids 400 -1447. Cells bearing the construct for the expression of Sgs1-(1-1119) yielded an insoluble protein that migrated with an apparent molecular mass of 160 kDa in a SDS-polyacrylamide gel. Expression of the other truncations yielded soluble prod-ucts that migrated with apparent molecular masses of 125 and 140 kDa, respectively, with the solubility of Sgs1-(400 -1268) substantially higher than that of Sgs1-(400 -1447). Because of its higher solubility, the Sgs1-(400 -1268) fragment was chosen for further studies. In the plasmid constructed for the expression of this fragment, pRB222, codons for MSTDPVYPYDVP-(amino acids 400 -1268 of Sgs1)-RRAVH 6 were placed downstream of a plasmid-borne inducible promoter of the yeast GAL1 gene. Codons for two peptide motifs, a hexapeptide epitope YPYDVP of flu virus HA and a stretch of six histidines, were added to the 5Ј-and 3Ј-end of the SGS1 sequence, respectively, to facilitate the detection and purification of the recombinant protein product (see "Experimental Procedures" for details). Cells of a protease-deficient S. cerevisiae strain were transformed with pRB222. Pilot experiments showed that when the transformed cells were grown and induced with galactose, a 125-kDa protein was overexpressed (Fig. 1C, lane b). Immunoblotting with an anti-HA monoclonal antibody confirmed that the overexpressed protein was the desired product (data not shown). Large scale preparations of the recombinant protein were then carried out, and the protein was purified to near homogeneity (Fig. 1C, lanes c-h).
DNA-dependent ATPase Activity-The purified recombinant Sgs1 fragment was tested for its ability to hydrolyze ATP in the presence and absence of DNA. As shown in Fig. 2, a DNA-dependent ATPase activity was readily detected. This activity co-purified with the peak of the recombinant Sgs1 protein when eluted from a heparin column (data not shown). Hydrolysis of ATP was equally efficient in the presence of several different DNA cofactors, including either single-or double-stranded Sgs1 DNA Helicase Activity of S. cerevisiae DNA (Fig. 2). In contrast, for the E. coli RecQ ATPase, it was previously shown that single-stranded DNA was a much more effective cofactor than double-stranded DNA (9). The slope of the beginning part of the plot shown in Fig. 2 corresponds to a rate of hydrolysis of approximately 10 ATP/s Sgs1 monomers.
DNA Binding by Sgs1-The DNA binding activity of the purified recombinant protein was investigated using the electrophoretic mobility shift assay (Fig. 3). The Sgs1 fragment was found to bind single-stranded circular X174 DNA (ssDNA), since the presence of increasing amounts of the protein progressively reduced the gel electrophoretic mobility of the DNA (Fig. 3, lanes a-f). Binding of the protein to supercoiled X174 appeared to be less efficient, and retardation of the DNA was observed only at the highest concentration of the recombinant protein used (lane l). The stable binding of the Sgs1 fragment to ssDNA did not require Mg(II) or nucleotide cofactors (data not shown).
To test the possibility that binding to the single-stranded DNA might be facilitated by secondary structures within it, Sgs1 binding to a synthetic duplex DNA with single-stranded tails at one end (the forked DNA substrate) was examined. The forked DNA was prepared by the annealing of two partially complementary oligonucleotides, 41 nucleotides in length. It was found that incubation of 32 P-labeled forked DNA with the Sgs1 fragment resulted in the formation of a stable protein-DNA complex (Fig. 4, lanes k-o), as detected by polyacrylamide gel electrophoresis. Under the same conditions, only very weak binding to either a single-stranded oligonucleotide (lanes a-e) or duplex DNA (lanes f-j) was observed.
Helicase Activity of the Recombinant Sgs1 Protein-To test for DNA helicase activity of the recombinant protein, we examined whether it could displace a 52-nucleotide 32 P-labeled oligodeoxynucleotide from single-stranded circular X174 DNA. The Sgs1 fragment was found to effect the dissociation of the oligonucleotide from the single-stranded DNA ring in the presence of ATP and Mg(II) (Fig. 5, lanes c-g, and lanes h and i).
Optimal activity occurred at a molar ratio of ATP to Mg(II) of 1:1 (data not shown). No activity was observed when ATP was replaced by the presumably nonhydrolyzable ATP analog ATP␥S (lane k). Furthermore, in the presence of 2 mM ATP, the addition of 1 mM ATP␥S to the reaction was found to significantly inhibit the unwinding activity (lane l). ATP could be replaced by dATP (lane m), but other common nucleotide triphosphates (e.g. GTP, TTP, and UTP) could not support DNA unwinding by the Sgs1 fragment (data not shown). These cofactor requirements are similar to those observed in the RecQmediated unwinding of DNA (9). Similar to the case of E. coli RecQ, the addition of Zn(II), but not Mn(II) or Ca(II), significantly inhibited the unwinding reaction (9).
A time course of the Sgs1-mediated strand displacement reaction is shown in Fig. 6. Over 50% of the labeled fragment was displaced within 5 min, and the reaction was close to completion within 10 min. The DNA unwinding activity was  a-e, f-j, and k-o, respectively). Protein-DNA complexes were analyzed by electrophoresis in a 6% nondenaturing polyacrylamide gel and autoradiography.
Sgs1 DNA Helicase Activity of S. cerevisiae very sensitive to the salt concentration (NaCl or potassium acetate) in the reaction buffer. There was a 50% decrease in the unwinding activity at a salt concentration between 30 and 40 mM; at 100 mM salt, the helicase activity was less than 10% of that seen in the low salt assay mixture (data not shown).
Unwinding of a DNA-RNA Hybrid-The recombinant Sgs1 fragment was also tested for its ability to unwind a DNA-RNA hybrid. A 26-nucleotide 32 P-labeled RNA or DNA oligomer was annealed to X174 virion DNA and incubated with the recombinant Sgs1 fragment in the presence of Mg(II) and ATP. Displacement of the oligoribonucleotide by the protein occurred with an efficiency similar to that of the oligodeoxynucleotide (data not shown). Like the DNA-DNA helicase activity, unwinding of the DNA-RNA hybrid was driven by the hydrolysis of ATP or dATP but not the other common nucleoside triphosphates (data not shown).
Polarity of the DNA Helicase-To determine the polarity of the helicase activity, the DNA substrates shown in Fig. 7A were used. These substrates were made by first annealing a 5Јor 3Ј-end-labeled oligonucleotide (66 nucleotides in length) with X174 ssDNA. The duplex region of each was then cut by PstI restriction endonuclease to yield a linear ssDNA with either a labeled complementary 40-mer or a labeled 26-mer bound to one of its ends. The recombinant Sgs1 fragment was found to efficiently displace the 5Ј-labeled 40-mer (substrate I; Fig. 7B). In contrast, unwinding and dissociation of the 3Ј-labeled 26mer by the protein was much less efficient (substrate II; Fig.  7B). The annealed 26-mer was not refractory to unwinding because of its sequence, since the original 66-mer was displaced with an efficiency close to that of the 40-mer (data not shown). These results indicate that the Sgs1 protein has a 3Ј to 5Ј polarity of unwinding with respect to the ssDNA flanking the duplex region, as was observed with E. coli RecQ helicase (9).
The Dependence of the Efficiency of Strand Displacement on the Length of the Displaced Strand-To investigate the dependence of the efficiency of DNA unwinding on the length of the duplex region, several DNA substrates were prepared using 32 P-labeled fragments of 52, 140, or 558 nucleotides. As depicted in Fig. 8A, each substrate was incubated with the indicated amounts of the recombinant Sgs1 fragment, and the  Sgs1 DNA Helicase Activity of S. cerevisiae products of the reaction were separated by polyacrylamide gel electrophoresis. It was found that the efficiency of strand displacement was markedly affected by its length, with more Sgs1 protein required to displace the longer DNA strands. Whereas the recombinant Sgs1 fragment displaced 60% of the 52-nucleotide fragment at a concentration of 1.2 nM (Fig. 8A, lane c, and  Fig. 8B), only 50% of the 140-nucleotide fragment and 5% of the 558-nucleotide fragment was displaced at a 4-fold higher enzyme concentration of 4.8 nM (see Fig. 8A, lanes i and n, and  Fig. 8B).
The effect of E. coli Ssb on the helicase activity of the recombinant Sgs1 protein was also examined. Using a DNA substrate carrying a 558-nucleotide 32 P-labeled strand, it was observed that the Sgs1 helicase activity was stimulated by the presence of increasing amounts of Ssb (Fig. 9). At the highest concentration of Ssb tested (70 nM), displacement of the 558-nucleotide DNA strand was enhanced by more than 12-fold. Up to 5-fold stimulation of Sgs1 helicase activity by Ssb was also observed using a 140-nucleotide strand annealed to its single-stranded template (data not shown). DISCUSSION We have shown that a purified recombinant Sgs1 fragment, which contains amino acid residues 400 -1268 of the full-length protein, possesses a helicase activity with partially duplex DNA substrates. The DNA unwinding reaction is dependent on the presence of ATP or dATP. Hydrolysis of the triphosphate appears to be obligatory, as ATP␥S can not substitute for ATP and inhibits the helicase activity in the presence of excess ATP. Unlike the E. coli RecQ helicase, which is stimulated by ssDNA but not double-stranded DNA, the hydrolysis of ATP by the recombinant Sgs1 protein is strongly stimulated by both forms of DNA. The polarity of unwinding is 3Ј to 5Ј with respect to the overhanging single-stranded DNA. The characteristics of the unwinding activity are similar to those of the E. coli RecQ protein (9), which shares homology with Sgs1 within the helicase domain. Of the other members of the RecQ family of DNA helicases (see Fig. 1A), the human RecQL protein has previously been purified and shown to have DNA unwinding activity in vitro (25,26). Recently, the Werner's syndrome and Bloom's syndrome proteins were also overexpressed and shown to possess a DNA helicase activity (27)(28)(29). It thus seems likely that all of the members of the RecQ family will share a common helicase function.
The efficiency of strand displacement by the recombinant Sgs1 fragment decreases sharply with increasing length of the strand to be displaced. The unwinding assay used here, however, detects only complete displacement events, and partially unwound fragments would not be scored. It is therefore plausible that with the longer strands reannealing of the DNA behind the translocating protein could reduce the observed efficiency of unwinding. Alternatively, the recombinant Sgs1 fragment might exhibit a low processivity in its reaction and might fall off the DNA before completing the displacement of the annealed fragment. The increased efficiency of unwinding of the 558-nucleotide fragment in the presence of E. coli Ssb protein is consistent with either of these interpretations.
We have also observed that Sgs1 has the ability to displace an RNA strand annealed to a longer DNA strand, with the same efficiency and cofactor requirements as displacing DNA from DNA. The product of the Werner's syndrome gene was also found to unwind RNA annealed to DNA (27). The ability to unwind RNA-DNA duplexes is not a common property of helicases. In one study, of four E. coli DNA helicases tested (30), only one (the UvrD protein) showed significant unwinding of RNA-DNA hybrids. It is not known whether the unwinding of RNA-DNA heteroduplex by the recombinant Sgs1 fragment is significant in terms of the physiological function of Sgs1 protein.
Using an assay based on the shift in the electrophoretic mobility of a DNA by bound protein, the recombinant Sgs1 protein fragment was shown to have a higher affinity for a forked DNA substrate than either single-or double-stranded DNA. This may have implications for the mechanism of DNA unwinding by Sgs1, since it has been suggested that preferential binding of the enzyme to such a junction might be important for helicase action (31). In addition, the high specificity for binding to a DNA junction could be important if the protein is to act as a decatenase in coordination with a DNA topoisomerase (see below).
The molecular mechanisms underpinning the physiological roles of the RecQ family of enzymes remain unclear. Because of the functional and physical association between yeast Sgs1 protein and DNA topoisomerase III, it was suggested that a complex between the two could act as a reverse gyrase to generate positive supercoiled regions in intracellular DNA (1). Whereas biochemically such a model is attractive, experimental data in support of such a possibility are lacking. Attempts to demonstrate a positive supercoiling activity of the recombinant Sgs1 fragment in the presence of yeast DNA topoisomerase III have been unsuccessful. 3 The absence of the N-and C-terminal regions of the full-length Sgs1 protein in the recombinant protein could, however, affect its formation of a complex with the topoisomerase or its interaction with DNA. An alternative model for the action of Sgs1 protein in vivo is that it might act by itself or jointly with DNA topoisomerase III in the unlinking of intertwined DNA strands. Such a decatenation activity might be involved near the end of DNA replication to resolve the intertwined parental DNA strands (1,5,32,33). Failure to resolve such structures could lead to DNA strand breakage and subsequently an increase in DNA recombination and chromosome missegregation. The Sgs1 protein-DNA topoisomerase III pair might also act directly on recombination intermediates to separate inadvertently paired DNA strands and thus reduce the mitotic recombination frequency (32,33). In the case of E. coli DNA topoisomerase III, which resembles yeast DNA topoisomerase III in its catalytic properties in vitro (33,34), the enzyme has been shown to be highly effective in the unlinking of parental strands in an in vitro plasmid DNA replication system (35,36).
The identification of the Bloom's and the Werner's syndrome determinants as homologues of the RecQ-type proteins in E. coli and yeasts has greatly stimulated interest in this class of enzymes. The functional and physical interaction between the yeast Sgs1 protein and yeast DNA topoisomerase III hints that their mammalian homologues might also interact. Interest-ingly, recent sequencing results suggest that there are two variants of mammalian DNA topoisomerase III encoded by genes located to chromosome 17p11.2-1 (37,38) and chromosome 22q11-12 (38). The plausible interactions between these variants, tentatively denoted DNA topoisomerases III␣ and III␤, and a group of helicases including the BLM, WRN, and RecQL proteins, offer challenging opportunities in their mechanistic and functional studies.