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• W2022027587 abstract "The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, ∼10 ± 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination. The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, ∼10 ± 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination. Rad54 is one of the key proteins of homologous recombination in eukaryotes (1Krogh B.O. Symington L.S. Annu. Rev. Genet. 2004; 38: 233-271Crossref PubMed Scopus (628) Google Scholar, 2Heyer W.D. Li X. Rolfsmeier M. Zhang X.P. Nucleic Acids Res. 2006; 34: 4115-4125Crossref PubMed Scopus (192) Google Scholar). Mutations in the RAD54 gene cause severe deficiency in homologous recombination and DNA repair (3Game J.C. Mutat. Res. 2000; 451: 277-293Crossref PubMed Scopus (62) Google Scholar, 4Wesoly J. Agarwal S. Sigurdsson S. Bussen W. Van Komen S. Qin J. van Steeg H. van Benthem J. Wassenaar E. Baarends W.M Ghazvini M. Tafel A.A. Heath H. Galjart N. Essers J. Grootegoed J.A. Arnheim N. Bezzubova O. Buerstedde J.M. Sung P. Kanaar R. Mol. Cell. Biol. 2006; 26: 976-989Crossref PubMed Scopus (115) Google Scholar). Structurally, Rad54 protein belongs to the Swi2/Snf2 family of DNA-dependent ATPases, whose members are best known for their chromatin remodeling activity (5Thoma N.H. Czyzewski B.K. Alexeev A.A. Mazin A.V. Kowalczykowski S.C. Pavletich N.P. Nat. Struct. Mol. Biol. 2005; 12: 350-356Crossref PubMed Scopus (155) Google Scholar). Biochemical studies have demonstrated a remarkable spectrum of Rad54 activities in vitro, indicating its multiple functions at various stages of homologous recombination (6Tan T.L. Kanaar R. Wyman C. DNA Repair. 2003; 2: 787-794Crossref PubMed Scopus (2) Google Scholar). Yeast and human Rad54 protein physically interacts with its cognate Rad51 protein (7Jiang H. Xie Y. Houston P. Stemke-Hale K. Mortensen U.H. Rothstein R. Kodadek T. J. Biol. Chem. 1996; 271: 33181-33186Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 8Golub E.I. Kovalenko O.V. Gupta R.C. Ward D.C. Radding C.M. Nucleic Acids Res. 1997; 25: 4106-4110Crossref PubMed Scopus (104) Google Scholar, 9Clever B. Interthal H. Schmuckli-Maurer J. King J. Sigrist M. Heyer W.D. EMBO J. 1997; 16: 2535-2544Crossref PubMed Scopus (156) Google Scholar) and stimulates the DNA pairing activity of Rad51, a key activity of homologous recombination (10Petukhova G. Stratton S. Sung P. Nature. 1998; 393: 91-94Crossref PubMed Scopus (345) Google Scholar, 11Mazina O.M. Mazin A.V. J. Biol. Chem. 2004; 279: 52042-52051Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Several mechanisms were proposed to account for this stimulation. At the presynaptic stage, Rad54 promotes binding of Rad51 to ssDNA 2The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; hRad54, human Rad54; BMH, bis-maleimidohexane; nt, nucleotide(s); PX-junction and -structure, partial X-junction and -structure, respectively. and stabilizes the Rad51-ssDNA filament in an ATP-independent manner (12Mazin A.V. Alexeev A.A. Kowalczykowski S.C. J. Biol. Chem. 2003; 278: 14029-14036Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Wolner B. van Komen S. Sung P. Peterson C.L. Mol. Cell. 2003; 12: 221-232Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 14Wolner B. Peterson C.L. J. Biol. Chem. 2005; 280: 10855-10860Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). At the synaptic stage, Rad54 stimulates DNA strand exchange and heteroduplex extension promoted by Rad51 through a different mechanism, dependent on ATP hydrolysis (10Petukhova G. Stratton S. Sung P. Nature. 1998; 393: 91-94Crossref PubMed Scopus (345) Google Scholar, 15Solinger J.A. Lutz G. Sugiyama T. Kowalczykowski S.C. Heyer W.D. J. Mol. Biol. 2001; 307: 1207-1221Crossref PubMed Scopus (96) Google Scholar). Rad54 forms a co-complex with the Rad51-ssDNA filament (12Mazin A.V. Alexeev A.A. Kowalczykowski S.C. J. Biol. Chem. 2003; 278: 14029-14036Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 16Mazin A.V. Bornarth C.J. Solinger J.A. Heyer W.D. Kowalczykowski S.C. Mol. Cell. 2000; 6: 583-592Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Within this co-complex, Rad54 is thought to be involved in interaction with the incoming dsDNA during the search for homology (11Mazina O.M. Mazin A.V. J. Biol. Chem. 2004; 279: 52042-52051Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Biochemical and single molecule experiments provided evidence that Rad54 protein can translocate along dsDNA using the energy of ATP hydrolysis (11Mazina O.M. Mazin A.V. J. Biol. Chem. 2004; 279: 52042-52051Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 17Van Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 18Ristic D. Wyman C. Paulusma C. Kanaar R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8454-8460Crossref PubMed Scopus (110) Google Scholar, 19Jaskelioff M. Van Komen S. Krebs J.E. Sung P. Peterson C.L. J. Biol. Chem. 2003; 278: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 20Amitani I. Baskin R.J. Kowalczykowski S.C. Mol. Cell. 2006; 23: 143-148Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). It was suggested that Rad54 promotes translocation of incoming dsDNA along the filament during the search for homology. In addition, it is likely that translocation of Rad54 and its meiosis-specific yeast homologue, Rdh54, on dsDNA is responsible for remodeling of nucleosomes (19Jaskelioff M. Van Komen S. Krebs J.E. Sung P. Peterson C.L. J. Biol. Chem. 2003; 278: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 21Alexeev A. Mazin A. Kowalczykowski S.C. Nat. Struct. Biol. 2003; 10: 182-186Crossref PubMed Scopus (192) Google Scholar, 22Alexiadis V. Kadonaga J.T. Genes Dev. 2002; 16: 2767-2771Crossref PubMed Scopus (132) Google Scholar) and removal of Rad51 from the dsDNA product at the terminal stage of DNA strand exchange (23Solinger J.A. Kiianitsa K. Heyer W.D. Mol. Cell. 2002; 10: 1175-1188Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 24Chi P. Kwon Y. Seong C. Epshtein A. Lam I. Sung P. Klein H.L. J. Biol. Chem. 2006; 281: 26268-26279Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Recently, we demonstrated that Rad54 protein binds with high specificity to branched DNA structures resembling Holliday junctions and promotes their branch migration in an ATPase-dependent manner (25Bugreev D.V. Mazina O.M. Mazin A.V. Nature. 2006; 442: 590-593Crossref PubMed Scopus (143) Google Scholar). hRad54 showed the strongest binding preference for the partial X-junction (PX-junction) in which one of the four arms contained ssDNA (Table 1). A lesser preference was seen for other branched DNA substrates, including forked DNA and the X-junction. Given the importance of the novel branch migration activity of Rad54, we set out to examine the basis for the observed binding specificity of hRad54 and to further characterize the mechanism of DNA binding.TABLE 1DNA substrates used for ATPase assays shown in Fig. 1a Numbers correspond to the oligonucleotides in supplemental Table S1. Open table in a new tab a Numbers correspond to the oligonucleotides in supplemental Table S1. Proteins—GST- and His6-tagged versions of hRad54 protein were expressed in Sf21 insect cells (11Mazina O.M. Mazin A.V. J. Biol. Chem. 2004; 279: 52042-52051Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). GST-hRad54 was purified as described previously (11Mazina O.M. Mazin A.V. J. Biol. Chem. 2004; 279: 52042-52051Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). His6-hRad54 purification was performed at 4 °C. Cells (10 g) stored at–80 °C were thawed and lysed by incubation in 10 volumes of ice-cold buffer A (50 mm Tris-HCl, pH 7.5, 200 mm KCl, 2 mm EDTA, 10% glycerol, 10 mm 2-mercaptoethanol, 0.5% Nonidet P-40) supplemented with EDTA-free protease inhibitors mixture (Roche Applied Science) for 30 min with constant stirring. The crude extract was clarified by centrifugation (100,000 × g for 60 min) (Fraction I) and loaded on a Q-Sepharose column (50 ml) equilibrated with T20 buffer (20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 10% glycerol, 10 mm 2-mercaptoethanol), containing 200 mm KCl. The flow-through fraction containing hRad54 (Fraction II) was diluted with K20 buffer (20 mm KH2PO4 (pH 7.5), 0.5 mm EDTA, 10% glycerol, 10 mm 2-mercaptoethanol) to 150 mm KCl and applied onto a 25-ml heparin column, which was developed with a 180-ml gradient from 150 to 1000 mm KCl in K20 buffer without EDTA. The hRad54 fractions (Fraction III; 35 ml; ∼300 mm KCl) were supplemented with 10 mm imidazole and loaded ona1mlof HisTrapHP column (GE Healthcare). The column was washed sequentially with K20 buffer without EDTA containing 500 mm KCl and 10, 20, and 30 mm imidazole. hRad54 protein was eluted with K20 buffer containing 500 mm KCl, 200 mm imidazole, and no EDTA. The eluate (Fraction IV; 2 ml), containing the bulk of the Rad54 protein, was further fractionated in a Superdex-200 column (58 ml), equilibrated with buffer K20 containing 500 mm KCl. hRad54 protein eluted from the S-200 column in a volume expected for a monomeric protein. The hRad54 eluate (Fraction V) was diluted with 4 volumes of K20, loaded on a Resource-S column (1 ml) equilibrated with buffer K20 containing 100 mm KCl, and eluted with a 20-ml gradient of KCl (100–600 mm) in K20 buffer. The fractions containing hRad54 protein were analyzed for nuclease contamination, pooled (Fraction VI), and stored in small aliquots at –80 °C. The protein appeared nearly homogeneous in a Coomassie-stained SDS-polyacrylamide gel. DNA—All oligonucleotides used in this study (supplemental Table S1) were purchased from Integrated DNA Technologies, Inc., in desalted form and further purified by electrophoresis in 6 –10% polyacrylamide gels containing 50% urea. The concentrations of the purified oligonucleotides were determined spectrophotometrically using extinction coefficients provided by the manufacturer. To prepare dsDNA or dsDNA with protruding ssDNA tails, complementary ssDNA oligonucleotides were annealed as described (26Bugreev D.V. Mazina O.M. Mazin A.V. Nature Prot. 2006; DOI:10.1038/nprot.2006.217Google Scholar) and stored at –20 °C. Oligonucleotides were labeled using [γ-32P]ATP and T4 polynucleotide kinase, as described previously (12Mazin A.V. Alexeev A.A. Kowalczykowski S.C. J. Biol. Chem. 2003; 278: 14029-14036Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). dsDNA fragments longer than a 135-mer were prepared by digestion of pUC19 plasmid DNA using the following restriction endonucleases (New England Biolabs): HaeIII to produce a 257-bp fragment; AluI to produce 521- and 679-bp fragments; ScaI and AflIII to produce a mixture of two fragments of 1373 and 1313 bp (designated as 1300 bp); and SmaI to produce a 2686-bp fragment (linear pUC19 dsDNA). 257, 521, and 679 dsDNA fragments were separated from the smaller fragments by a 6% nondenaturing PAGE and recovered as described (26Bugreev D.V. Mazina O.M. Mazin A.V. Nature Prot. 2006; DOI:10.1038/nprot.2006.217Google Scholar). 1300- and 2686-bp DNA fragments were prepared by phenol extraction followed by ethanol precipitation. ATPase Assay—The hydrolysis of ATP by Rad54 protein was monitored spectrophotometrically as described previously (27Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Crossref PubMed Scopus (200) Google Scholar). The oxidation of NADH, coupled to ADP phosphorylation, resulted in a decrease in absorbance at 340 nm, which was continuously monitored by a Hewlett-Packard 8453 diode array spectrophotometer using UV-visible ChemStation software. The rate of ATP hydrolysis was calculated from the rate of change in absorbance using the following formula: rate of A340 decrease (s–1) × 9880 = rate of ATP hydrolysis (μm/min). The reactions in standard buffer containing 25 mm Tris acetate, pH 7.5, 10 mm magnesium acetate (unless indicated otherwise), 1 mm dithiothreitol, 2 mm ATP, 3 mm phosphoenolpyruvate, pyruvate kinase (20 units/ml), lactate dehydrogenase (20 units/ml), NADH (200 μg/ml), and the indicated concentrations of Rad54 protein and DNA were carried out at 30 °C. In preliminary experiments, it was determined that 2 mm was a saturating concentration of ATP for all DNA substrates used in this study. Kinetic parameters of these reactions were determined using GraphPad Prism 4.03 software. Concentrations of free Mg2+ ion were calculated using WebMaxC2.10 (available on the World Wide Web) applying the following parameters: temperature, 30 °C; pH 7.5; and ionic strength, 0.05 (28Schmitz C. Perraud A.L. Johnson C.O. Inabe K. Smith M.K. Penner R. Kurosaki T. Fleig A. Scharenberg A.M. Cell. 2003; 114: 191-200Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). hRad54 Protein Cross-linking—In the standard cross-linking reaction, His6 tag hRad54 (850 nm) was preincubated with DNA (0.4 or 4 μm, molecules) for 5 min at 25 °C in 10 μl of reaction buffer containing 20 mm HEPES-KOH, pH 7.5, 5 mm EDTA, 5 mm MgCl2, 50mm NaCl, followed by the addition of 2 μlof bis-maleimidohexane (BMH) (Pierce) to a final concentration of 25 or 100 μm. After a 10-min incubation at 25 °C, the reaction was quenched by the addition of 1 μl of 14.3 m 2-mercaptoethanol. The samples were analyzed by SDS-PAGE in 7.5% gels. The hRad54 protein was visualized by silver staining (Invitrogen). Branch Migration Assay—The hRad54 protein (100 nm, unless indicated otherwise) was incubated with 32P-labeled synthetic PX-junction (number 71/169/170/171) (33 nm, molecules), 32P-labeled synthetic X-junction (number 71/170/234/235) (33 nm, molecules), or 32P-labeled synthetic PX-junction (number 265/266/269/270) (20 or 30 nm, molecules) in a 90-μl branch migration buffer containing 25 mm Tris acetate, pH 7.5, 2mm ATP, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin, the ATP-regenerating system (10 units/ml creatine phosphokinase and 15 mm creatine phosphate), and the indicated concentrations of magnesium acetate. The reactions were carried out at 30 °C or in the case of PX-junction (number 265/266/269/270) at 20 °C. Aliquots (10 μl) were withdrawn, and DNA substrates were deproteinized by treatment with stop buffer (1.4% SDS, 960 μg/ml proteinase K, 7.5% glycerol, 0.015% bromphenol blue) for 5 min at 22 °C. Samples were analyzed by electrophoresis in 8% polyacrylamide gels (29:1) in 1× TBE buffer (90 mm Tris borate, pH 8.3, and 1 mm EDTA) at 22 and at 4 °C for PX- and X-structure, respectively. The gels were dried on DE81 chromatography paper (Whatman) and quantified using a Storm 840 PhosphorImager (Amersham Biosciences). In hRad54 stoichiometric titrations, the initial rates of branch migration were determined using branch migration buffer containing 3 mm magnesium acetate. The reactions were carried out for 5 min at 20 °C. Time points were taken at 1, 3, and 5 min for each protein concentration tested. The initial rate was determined using the linear part of the kinetic curve during the first 3 min of the reaction. In some experiments, the ATPase regeneration system containing 3 mm phosphoenolpyruvate, pyruvate kinase (20 units/ml), lactate dehydrogenase (20 units/ml), and NADH (200 μg/ml) was used instead of the ATP regeneration system containing creatine phosphokinase and creatine phosphate, with no apparent effect on the initial rate measurements. hRad54 Shows Binding Preferences for Branched DNA substrates with ssDNA Arms—Previously, we found that hRad54 shows DNA binding preference for branched DNA substrates (25Bugreev D.V. Mazina O.M. Mazin A.V. Nature. 2006; 442: 590-593Crossref PubMed Scopus (143) Google Scholar). hRad54 possesses a dsDNA-dependent ATPase activity, which is essential for DNA branch migration and DNA translocation. Here, using the ATPase activity as a readout, we further characterized hRad54 DNA substrate specificity for a broad range of DNA substrates. We measured the initial rate of ATP hydrolysis of hRad54 protein in the presence of various branched oligonucleotide-derived DNA substrates over a range of DNA concentrations (Fig. 1). The results demonstrate that branched DNA structures (Table 1) with one single-stranded arm (PX-junction, 3′-flap, 5′-flap) stimulate the velocity (Vmax) of hRad54 ATP hydrolysis ∼1.5–2.0-fold greater than branched DNA substrates with fully dsDNA arms (X-junction, replication fork) or with two ssDNA arms (Kappa and forked DNA). The 3′-flap and 5′-flap structures containing two dsDNA arms and one ssDNA arm were nearly as efficient in stimulating the ATPase activity of hRad54 protein as the PX-junction, which was the best substrate among the previously examined structures (25Bugreev D.V. Mazina O.M. Mazin A.V. Nature. 2006; 442: 590-593Crossref PubMed Scopus (143) Google Scholar). Overall, our results show the following order of preference for DNA substrates according to their proficiency in inducing hRad54 ATPase: PX-structure ≥ 3′-flap ≥ 5′-flap >> Kappa ≥ X-structure = replication fork > forked DNA > dsDNA. Previously, it was shown that ssDNA supports the ATPase activity of hRad54 poorly (29Swagemakers S.M. Essers J. de Wit J. Hoeijmakers J.H. Kanaar R. J. Biol. Chem. 1998; 273: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Structural Requirements for Branched DNA Molecules That Serve as Substrates for hRad54—To gain a better understanding of how Rad54 protein interacts with branched DNA substrates, we determined the minimal length of ssDNA and dsDNA regions of branched DNA required for efficient hRad54 binding and stimulation of its ATPase activity. Starting from linear dsDNA, we gradually increased the complexity of DNA substrates by constructing additional DNA branches and varying the length of the branches. To determine the minimal length of linear dsDNA that supports the ATPase activity of Rad54, we measured the initial rate of ATP hydrolysis by hRad54 as a function of dsDNA concentration for each of a set of dsDNA fragments with lengths varying from 17 to 2686 bp (see examples in supplemental Fig. S1). From the obtained data, we determined the KM for each dsDNA fragment (K DNAm) (Fig. 2A and Table 2). The KM value decreases sharply from 61.6 to 0.62 μm with an increase of dsDNA length from 17 to 63 bp. The KM decreases slightly to 0.44 μm for 90 bp dsDNA fragment and levels out within a 0.3–0.4 μm range for dsDNA fragments up to 2686 bp long. At the same time, the velocity of ATP hydrolysis increases sharply with an increase of dsDNA length from 17 to 63–90 bp and then less steeply until it plateaus at 1300 bp (Fig. 2B). It was observed previously for T4 gp41 helicase that a sharp decrease in the K DNAm and concomitant increase in the ATPase Vmax occurs when the size of DNA approaches the DNA binding site size of the protein (30Young M.C. Schultz D.E. Ring D. von Hippel P.H. J. Mol. Biol. 1994; 235: 1447-1458Crossref PubMed Scopus (76) Google Scholar). By this estimate, the apparent size of the hRad54 DNA binding site required for basal ATPase activity is between 63 and 90 bp. Based on the model of Peter von Hippel and co-workers (30Young M.C. Schultz D.E. Ring D. von Hippel P.H. J. Mol. Biol. 1994; 235: 1447-1458Crossref PubMed Scopus (76) Google Scholar), the second phase of increase in the velocity of ATP hydrolysis for dsDNA fragments from 63–90 bp to 1300 bp at a constant K DNAm value was consistent with translocation of hRad54 protein along the dsDNA (11Mazina O.M. Mazin A.V. J. Biol. Chem. 2004; 279: 52042-52051Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 30Young M.C. Schultz D.E. Ring D. von Hippel P.H. J. Mol. Biol. 1994; 235: 1447-1458Crossref PubMed Scopus (76) Google Scholar). The Vmax data for these DNA fragments have been fit to an equation hyperbolic in DNA length (Fig. 2B). The DNA length at half-plateau height, 280 ± 30 bp, indicates the average translocation distances for hRad54 under tested conditions.TABLE 2The KMDNA of the ATPase activity of hRad54 as a function of the length of dsDNA substratesDNA lengthKMDNAOligonucleotidesaNumbers correspond to the oligonucleotides in supplemental Table S1.bpμm (nt)1762 ± 2917/182510 ± 1.4112/113324.3 ± 0.65/6483.4 ± 0.425/26630.62 ± 0.41/2900.44 ± 0.390/911350.40 ± 0.2114/11513000.38 ± 0.1pUC19 fragment26860.42 ± 0.1Linear pUC19a Numbers correspond to the oligonucleotides in supplemental Table S1. Open table in a new tab To examine whether an ssDNA tail attached to a short dsDNA fragment can enhance the ATPase activity of Rad54 protein, we constructed a set of tailed DNA molecules containing an invariable 32-bp dsDNA region with ssDNA oligo(dT) tails of various lengths. An oligo(dT) sequence was chosen, because it does not support the ATPase activity of Rad54; in contrast, ssDNA sequences of mixed base composition support some small level of ATP hydrolysis (29Swagemakers S.M. Essers J. de Wit J. Hoeijmakers J.H. Kanaar R. J. Biol. Chem. 1998; 273: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). 3O. M. Mazina and A. V. Mazin, unpublished data. We found that the velocity of ATP hydrolysis by hRad54 protein was increased by the addition of ssDNA tails to a 32-bp dsDNA fragment. The increase was dependent on the length of ssDNA tails, reaching a maximum, ∼4-fold stimulation, with an ssDNA tail of 45–60 nt (dT45 and dT60) (Table 3). The polarity of ssDNA tails (3′ to 5′ or 5′ to 3′) relative to the duplex DNA had no effect on the ATPase activity of hRad54 (Table 3). The presence of ssDNA in the DNA substrate appeared to be important for stimulation of the hRad54 ATPase activity, because in trans dT47 ssDNA inhibited dsDNA-dependent ATPase activity of hRad54 (data not shown). Previously, it was shown that ssDNA inhibits the ATPase activity of yeast Rad54. 4S. Kowalczykowski and C. Bornarth, personal communication. In contrast to a 32-bp dsDNA fragment, the addition of 32- or 45-nt oligo(dT) ssDNA tails to longer dsDNA fragments (62 or 90 bp) did not lead to a noticeable stimulation of ATP hydrolysis (Fig. 3). We suggested that ssDNA tails stimulate the ATPase activity of hRad54 mainly by increasing the overall length of short DNA substrates up to the DNA binding site size of hRad54. The obtained value for this site, 80–90 nt/bp, agrees with the estimate reported above for dsDNA fragments.TABLE 3ATPase activity of hRad54 with various DNA substrates of ascending complexitya For each DNA substrate, the rates of ATP hydrolysis were determined in a broad range of DNA concentrations up to saturation (see supplemental Fig. 1). hRad54 concentration was 60 nm. The kcat values were calculated by fitting the data to the Michaelis-Menten equation.b Numbers correspond to the oligonucleotides in supplemental Table S1. Open table in a new tab a For each DNA substrate, the rates of ATP hydrolysis were determined in a broad range of DNA concentrations up to saturation (see supplemental Fig. 1). hRad54 concentration was 60 nm. The kcat values were calculated by fitting the data to the Michaelis-Menten equation. b Numbers correspond to the oligonucleotides in supplemental Table S1. We further increased the complexity of DNA substrates by adding a second ssDNA arm to dT45-tailed DNA, creating forked DNA (Table 3). We investigated the effect of the length of this ssDNA arm on the ATPase activity of hRad54. We found that forked DNA stimulated the ATPase activity of hRad54 stronger than tailed DNA (Table 3). The highest stimulation, ∼2.5-fold over tailed DNA, was observed when the length of the second ssDNA arm was between 30 and 45 nt. Next, using the optimal forked DNA substrate, containing two dT arms of 45 and 30 nt, we determined the minimal length of the dsDNA branch required for the ATPase activity of hRad54 protein. The results showed that the dsDNA branch can be decreased to 12 bp without significant loss of the ATPase activity (Table 3). However, shortening of the dsDNA region to 8 bp decreased the velocity of ATP hydrolysis dramatically, ∼4-fold. The calculated melting temperature of the 8-bp duplex was 35.1 °C; therefore, it might become unstable at the reaction temperature (30 °C). However, even at 20 °C, forked DNA with a 12-bp dsDNA branch was a far superior substrate for Rad54 ATPase activity than that with an 8-bp dsDNA branch (data not shown). Thus, a 12-bp dsDNA branch in forked DNA substrate was sufficient for stimulation of ATP hydrolysis by hRad54. Shortening of the dsDNA region to 2 bp decreases the hRad54 ATPase activity to a very low level (7-fold lower than for an 8-bp dsDNA region), typically observed in the presence of ssDNA. We then constructed a set of 3′-flap DNA molecules with a 45-nt ssDNA arm (dT45 or mixed base composition) and two dsDNA arms (mixed base composition) of variable length (Table 3). When the length of both dsDNA arms was 15 bp, the ATPase activity (Vmax) was ∼1.6-fold higher than for the best forked DNA substrate (Table 3). The increase in length of dsDNA arms to 25, 37, or 45 bp did not increase the ATPase activity of hRad54 protein further or even slightly decreased it when the dsDNA arms were 45 and 32 bp. The sequence of the ssDNA arm in the flapped DNA was not apparently essential for the ATPase activity, since replacement of the dT45 arm with the ssDNA arm of mixed base composition did not significantly affect the rate of ATP hydrolysis. The decrease in length of ssDNA arm to 30 and 15 nt caused a decrease in the rate of ATP hydrolysis by hRad54 (data not shown). Finally, we constructed the PX-junction containing three dsDNA branches of 15 bp each and an ssDNA arm of 45 nt. The rate of ATP hydrolysis with this substrate appeared to be approximately the same as with the flap DNA described above (Table 3). Thus, in the course of analysis of a series of DNA substrates of ascending complexity, the flap DNA containing two 15-bp dsDNA arms and a 45-nt ssDNA arm, emerged as the minimal substrate that supports optimal Rad54 ATPase activity, about 15-fold better than 32-bp linear dsDNA. Henceforth we refer it as the “minimal flap DNA substrate.” The Stoichiometry of hRad54 Binding to the Minimal Flap DNA Substrate—Although on a gel filtration column, Rad54 protein elutes as a monomer (31Petukhova G. Van Komen S. Vergano S. Klein H. Sung P. J. Biol. Chem. 1999; 274: 29453-29462Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar)3 the stoichiometry of Rad54-DNA complexes is still unknown. Here we measured the stoichiometry of hRad54 binding to the minimal flap DNA substrate. For this purpose, we measured the rate of ATP hydrolysis as a function of (i) DNA concentration at fixed hRad54 protein concentration (60 nm) and (ii) hRad54 protein concentration at fixed DNA concentration (30 nm, molecules). Since the GST domain is known for its ability to dimerize in solution (32Tudyka T. Skerra A. Protein Sci. 1997; 6: 2180-2187Crossref PubMed Scopus (65) Google Scholar), we performed these experiments with both GST-Rad54 and His6-Rad54 proteins to exclude any possible effect of such dimerization. The results, however, appeared to be identical for both versions of hRad54. Titration" @default.
• W2022027587 created "2016-06-24" @default.
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• W2022027587 creator A5023103944 @default.
• W2022027587 creator A5071817184 @default.
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• W2022027587 date "2007-07-01" @default.
• W2022027587 modified "2023-10-18" @default.
• W2022027587 title "Interactions of Human Rad54 Protein with Branched DNA Molecules" @default.
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