Equilibrium Binding Assays Reveal the Elevated Stoichiometry and Salt Dependence of the Interaction between Full-length Human Sex-determining Region on the Y Chromosome (SRY) and DNA
2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês
10.1074/jbc.m112366200
ISSN1083-351X
AutoresStéphanie Baud, Emmanuel Margeat, Serge Lumbroso, Françoise Paris, Charles Sultan, Catherine A. Royer, Nicolas Poujol,
Tópico(s)Animal Genetics and Reproduction
ResumoIn an effort to better define the molecular mechanism of the functional specificity of human sex-determining region on the Y chromosome (SRY), we have carried out equilibrium binding assays to study the interaction of the full-length bacterial-expressed protein with a DNA response element derived from the CD3ε gene enhancer. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the protein to the labeled target DNA. Our results indicate that the full-length human wild-type SRY (SRYWT) forms a complex of high stoichiometry with its target DNA. Moreover, we have demonstrated a strong salt dependence of both the affinity and specificity of the interaction. We have also addressed the DNA bending properties of full-length human SRYWT in solution by fluorescence resonance energy transfer and revealed that maximal bending is achieved with a protein to DNA ratio significantly higher than the classical 1:1. Oligomerization thus appears, at least in vitro, to be tightly coupled to SRY-DNA interactions. Alteration of protein-protein interactions observed for the mutant protein SRYY129N, identified in a patient presenting with 46,XY sex reversal, suggests that oligomerization may play an important role in vivo as well. In an effort to better define the molecular mechanism of the functional specificity of human sex-determining region on the Y chromosome (SRY), we have carried out equilibrium binding assays to study the interaction of the full-length bacterial-expressed protein with a DNA response element derived from the CD3ε gene enhancer. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the protein to the labeled target DNA. Our results indicate that the full-length human wild-type SRY (SRYWT) forms a complex of high stoichiometry with its target DNA. Moreover, we have demonstrated a strong salt dependence of both the affinity and specificity of the interaction. We have also addressed the DNA bending properties of full-length human SRYWT in solution by fluorescence resonance energy transfer and revealed that maximal bending is achieved with a protein to DNA ratio significantly higher than the classical 1:1. Oligomerization thus appears, at least in vitro, to be tightly coupled to SRY-DNA interactions. Alteration of protein-protein interactions observed for the mutant protein SRYY129N, identified in a patient presenting with 46,XY sex reversal, suggests that oligomerization may play an important role in vivo as well. Sex-determining region on the Y chromosome (SRY) 1The abbreviations used are: SRYsex-determining region on the Y chromosomeFRETfluorescence resonance energy transferHMGhigh mobility groupHPLChigh pressure liquid chromatography 1The abbreviations used are: SRYsex-determining region on the Y chromosomeFRETfluorescence resonance energy transferHMGhigh mobility groupHPLChigh pressure liquid chromatography is the master genetic switch that triggers development of the bipotential gonad into testes in mammalian embryos (1Sinclair A.H. Berta P. Palmer M.S. Hawkins J.R. Griffiths B.L. Smith M.J. Foster J.W. Frischauf A.M. Lovell-Badge R. Goodfellow P.N. Nature. 1990; 346: 240-244Crossref PubMed Scopus (2571) Google Scholar, 2Berta P. Hawkins J.R. Sinclair A.H. Taylor A. Griffiths B.L. Goodfellow P.N. Fellous M. Nature. 1990; 348: 448-450Crossref PubMed Scopus (428) Google Scholar). The protein it encodes is a member of a large family of nuclear proteins harboring a 79-amino acid motif known as a high mobility group (HMG) box (3Baxevanis A.D. Landsman D. Nucleic Acids Res. 1995; 23: 1604-1613Crossref PubMed Scopus (187) Google Scholar). HMG box-containing proteins can be classified into two major groups based on the degree of sequence specificity in DNA binding and the number of HMG boxes within a protein. One group includes UBF, HMG-1 and MT-TF1, which have multiple HMG boxes and recognize DNA with low or no specificity. The other group includes transcriptional regulators such as LEF-1 and Sox (SRY-box related) proteins, including SRY, that possess a single HMG box and show sequence-specific DNA binding. Thus, the SRY protein is a DNA-binding protein that recognizes certain AT-rich sequences (4Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (375) Google Scholar, 5Haqq C.M. King C.Y. Donahoe P.K. Weiss M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1097-1101Crossref PubMed Scopus (140) Google Scholar, 6Giese K. Pagel J. Grosschedl R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3368-3372Crossref PubMed Scopus (107) Google Scholar) including the consensus binding sequence A/TAACAAAT/A obtained by random site selection (7Harley V.R. Goodfellow P.N. Mol. Reprod. Dev. 1994; 39: 184-193Crossref PubMed Scopus (82) Google Scholar). Upon binding, human SRYWTinduces a 60–83° bend in the DNA helix as demonstrated by circular permutation assays (6Giese K. Pagel J. Grosschedl R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3368-3372Crossref PubMed Scopus (107) Google Scholar, 8Ferrari S. Harley V.R. Pontiggia A. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1992; 11: 4497-4506Crossref PubMed Scopus (380) Google Scholar, 9Pontiggia A. Rimini R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (255) Google Scholar), NMR structure (10Werner M.H. Huth J.R. Gronenborn A.M. Clore G.M. Cell. 1995; 81: 705-714Abstract Full Text PDF PubMed Scopus (431) Google Scholar), and fluorescence resonance energy transfer (11Ukiyama E. Jancso-Radek A., Li, B. Milos L. Zhang W. Phillips N.B. Morikawa N. King C.Y. Chan G. Haqq C.M. Radek J.T. Poulat F. Donahoe P.K. Weiss M.A. Mol. Endocrinol. 2001; 15: 363-377Crossref PubMed Scopus (29) Google Scholar). Both DNA binding and bending capacities were demonstrated as essential for testis development on the basis of the study of the biochemical consequences of SRY mutations (4Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (375) Google Scholar, 9Pontiggia A. Rimini R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (255) Google Scholar, 12Nasrin N. Buggs C. Kong X.F. Carnazza J. Goebl M. Alexander-Bridges M. Nature. 1991; 354: 317-320Crossref PubMed Scopus (152) Google Scholar). To date, 36 SRY mutations have been reported (13Schaffler A. Barth N. Winkler K. Zietz B. Rummele P. Knuchel R. Scholmerich J. Palitzsch K.D. J. Clin. Endocrinol. Metab. 2000; 85: 2287-2292Crossref PubMed Scopus (21) Google Scholar) in patients with gonadal dysgenesis/XY sex reversal, and the large majority (33 of 36) of the patients were phenotypically normal 46,XY females with complete gonadal dysgenesis. The strong bending of DNA together with the lack of a potential trans-regulation domain in human SRY has led to the suggestion that the protein may modulate transcription by acting architecturally in the assembly of a nucleoprotein complex (9Pontiggia A. Rimini R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (255) Google Scholar). However, despite the critical role of SRY in the cascade of gene regulation leading to maleness, the direct targets of SRY remain to be positively identified. sex-determining region on the Y chromosome fluorescence resonance energy transfer high mobility group high pressure liquid chromatography sex-determining region on the Y chromosome fluorescence resonance energy transfer high mobility group high pressure liquid chromatography Because the first step in such a cascade is DNA recognition, a thorough, quantitative understanding of the structure-energetic function relations in this system is essential. A number of studies of the interactions between SRY and duplex DNA, all using mobility shift assays, have been published (8Ferrari S. Harley V.R. Pontiggia A. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1992; 11: 4497-4506Crossref PubMed Scopus (380) Google Scholar, 9Pontiggia A. Rimini R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (255) Google Scholar, 14Peters R. King C.Y. Ukiyama E. Falsafi S. Donahoe P.K. Weiss M.A. Biochemistry. 1995; 34: 4569-4576Crossref PubMed Scopus (42) Google Scholar, 15Trimmer E.E. Zamble D.B. Lippard S.J. Essigmann J.M. Biochemistry. 1998; 37: 352-362Crossref PubMed Scopus (84) Google Scholar). For example, Ferrari et al. (8Ferrari S. Harley V.R. Pontiggia A. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1992; 11: 4497-4506Crossref PubMed Scopus (380) Google Scholar) examined SRY-DNA interactions, but their study was restricted to a construct containing only the HMG box. Similar studies using wild-type and mutant SRY isolated from complete gonadal dysgenesis have been performed but were still restricted to the SRY HMG box (9Pontiggia A. Rimini R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (255) Google Scholar). However, domains of SRY distinct from the HMG box have been implicated in the modulation of DNA binding properties (16Desclozeaux M. Poulat F. de Santa Barbara P. Soullier S. Jay P. Berta P. Boizet-Bonhoure B. Biochim. Biophys. Acta. 1998; 1397: 247-252Crossref PubMed Scopus (29) Google Scholar). Trimmer et al. (15Trimmer E.E. Zamble D.B. Lippard S.J. Essigmann J.M. Biochemistry. 1998; 37: 352-362Crossref PubMed Scopus (84) Google Scholar) have reported studies of full-length human SRY and human SRY-HMG box domain interactions with a 20-bp DNA oligonucleotide. Their results suggested that the affinities of both constructs were in the nanomolar range but also pointed out sharp binding transitions and higher order complexes with multiples sites on the probes that precluded of a thermodynamic analysis with confidence. The gel mobility shift method provides interesting information concerning the number of stoichiometric complexes formed but suffers from its nonequilibrium nature and the relatively large signal to noise ratio inherent in the titration curves derived from quantification of the bands. The quality of such data is usually insufficient for determination of the presence and degree of cooperativity in binding. In the present work, we have used a fluorescence-based binding assay to quantitatively characterize the interaction between full-length bacterial-expressed human SRY and its target DNA. Our equilibrium assays are based on the observations of changes in the fluorescence anisotropy of a fluorescein-labeled DNA target upon binding by the protein. Because rotational diffusion of the free oligonucleotide is quite rapid, the anisotropy of the fluorescent dye covalently bound to the oligonucleotide is quite low, i.e. little orientation of the polarized exciting light is retained in the emission. However, because binding by the protein significantly slows the rotational diffusion of the oligonucleotide, much more of the exciting light polarization is retained in the emission. These experiments can be performed with very low concentrations of target DNA and provide data of very high precision and reproducibility (17Grillo A.O. Brown M.P. Royer C.A. J. Mol. Biol. 1999; 287: 539-554Crossref PubMed Scopus (40) Google Scholar, 18Boyer M. Poujol N. Margeat E. Royer C.A. Nucleic Acids Res. 2000; 28: 2494-2502Crossref PubMed Scopus (65) Google Scholar, 19Margeat E. Poujol N. Boulahtouf A. Chen Y. Muller J.D. Gratton E. Cavailles V. Royer C.A. J. Mol. Biol. 2001; 306: 433-442Crossref PubMed Scopus (61) Google Scholar). Thus, they can be used to quantitatively characterize the affinity, cooperativity, and, eventually, the kinetics of biomolecular interactions. To gain further insight into the molecular basis for SRY function, we performed binding experiments with a rare SRY mutant, identified in partial rather than complete gonadal dysgenesis, that may present more subtle biochemical consequences and thus be more difficult to reveal. Only three SRY mutations have been reported to date with this partial clinical presentation, and all of them were located outside the HMG box (20Brown S., Yu, C. Lanzano P. Heller D. Thomas L. Warburton D. Kitajewski J. Stadtmauer L. Am. J. Hum. Genet. 1998; 62: 189-192Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 21Domenice S. Yumie Nishi M. Correia Billerbeck A.E. Latronico A.C. Aparecida Medeiros M. Russell A.J. Vass K. Marino Carvalho F. Costa Frade E.M. Prado Arnhold I.J. Bilharinho Mendonca B. Hum. Genet. 1998; 102: 213-215Crossref PubMed Scopus (60) Google Scholar, 22McElreavey K. Vilain E. Barbaux S. Fuqua J.S. Fechner P.Y. Souleyreau N. Doco-Fenzy M. Gabriel R. Quereux C. Fellous M. Berkovitz G.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8590-8594Crossref PubMed Scopus (60) Google Scholar). In contrast, the fourth such mutation (Y129N) examined here is located at the C-terminal end of the HMG box. We have used this anisotropy-based assay to evaluate the affinity, specificity, and cooperativity of the interaction between the full-length wild-type and mutant human SRY with the consensus target DNA at various salt concentrations. We also addressed the DNA bending properties of both full-length human SRYs in solution by fluorescence resonance energy transfer (FRET). His-tagged proteins were overexpressed in Escherichia coli and purified by nickel-nitrilotriacetic acid-agarose beads (Qiagen, Courtaboeuf, France) under denaturing conditions. DNA encoding for the full-length SRY protein was amplified from genomic DNA extracted from a fertile subject and from the patient presenting with the Y129N substitution. For this purpose, the oligonucleotides SRYs (5′-TCACGGATCCATGCAATCATATGCTTCTG-3′) and SRYas (5′-CTAATTAAGCTTCAGCTTTGTCCAGTGGC-3′) were purchased from Genosys (Montigny-le-Bretonneux, France). Reaction products and expression plasmid pQE30 (Qiagen) were cleaved with BamHI and Hin dIII, purified, ligated, and cloned into E. coli XL1. The resulting plasmids pQE30-SRYWT and pQE30-SRYY129N were checked by sequencing. The plasmids were introduced into E. coli SG13009-rep4 (Qiagen). The cells were grown in LB medium with 100 μg/ml ampicillin and 25 μg/ml neomycin at 37 °C and induced at an A600 of 0.7 with 0.5 mmisopropyl β-d-thiogalactopyranoside. After 2h, the cells were harvested by centrifugation and lysed under denaturing conditions (6 m guanidine hydrochloride, 20 mm Tris-HCl, pH 8.0, 5 mm imidazole, 500 mm NaCl, and 5 mm β-mercaptoethanol). The cell lysates were passed over a nickel-nitrilotriacetic acid-agarose column (Qiagen), and the SRY proteins were eluted according to the manufacturer's recommendations (6 m urea, 20 mm bis-Tris-HCl, pH 5.0, 200 mm imidazole, 500 mm NaCl, and 5 mmβ-mercaptoethanol). The unfolded protein was subject to a concentration under nitrogen (Amicon, YM10) and then added dropwise to the renaturing buffer (10 mm Tris-HCl, pH 7.0, 150 mm NaCl, 1 mm EDTA, 5 mmdithiothreitol, and 10% glycerol) at 0 °C on ice. The folded proteins were then loaded on a fast protein liquid chromatography Superdex 75 size exclusion column (AKTA prime; Amersham Biosciences). The proteins eluted at the expected monomer molecular mass were homogeneous by Coomassie Blue staining of a SDS-polyacrylamide gel, and their respective concentrations were calculated by using the extinction coefficient (34640 m −1 cm−1 at 280 nm). Oligonucleotides were purchased in HPLC-purified form from Genset S.A. (Paris, France). The fluorescein and rhodamine X labels were incorporated by the supplier using phosphoramidite chemistry, and all the free probe was thus eliminated in the synthesizer and subsequent HPLC purification. The labeling ratios for the oligonucleotides were 60% and 99% for fluorescein- and rhodamine X-labeled oligonucleotides, respectively. The sense and antisense strands were annealed by heating a 1:1 molar ratio of unlabeled antisense with fluorescein-labeled sense strands to 85 °C for 5 min and slowly cooling them in a thermocycler (Gen-amp 2400; PerkinElmer Life Sciences), resulting in a duplex probe used for the anisotropy assays (F-SRBE). A double-labeled duplex probe (F-SRBE-R) intended for FRET was similarly prepared, except that the antisense strand was rhodamine X-labeled. The 23-bp probe referred to here as SRBE has the sequence given below for the sense strand : 5′-CCCTGCAGGTAACAATGTCGGCT-3′. Binding assays were performed using a Beacon 2000 polarization instrument (Panvera Corp., Madison, WI) regulated at 4 °C. Each point in the titration curves was obtained by starting with 200 μl of a concentrated solution of SRY and 5 nm F-SRBE. Aliquots of 40 μl were successively removed from the starter solution and replaced by 40 μl containing 5 nm F-SRBE. The buffer solution was 10 mmTris-HCl, 1 mm EDTA, 10% glycerol, 1 mmdithiothreitol, pH 7.5 (TEGD buffer) and contained the indicated concentration of KCl. Tubes were equilibrated at 4 °C for 5 min before measurement, and the anisotropy was measured successively until stabilized. The reported values are the average of five to seven measurements after stabilization. Anisotropy is calculated as the ratio of the difference between vertical and horizontal emission intensities (I// and I⊥) normalized to the total intensity: A = (I// − I⊥)/(I// + 2I⊥) Time-resolved fluorescence experiments were performed in the frequency domain using ISS frequency domain acquisition electronics (ISS Inc., Champaign, IL). The excitation light was at 450 nm from the frequency doubled mode-locked output of a Spectra Physics Tsunami Titanium-Saphir laser excited with the light of a Millenia X diode-pumped laser. Pulse width was 2 ps at 4 MHz, and the frequency response was measured at harmonic frequencies from 4 to 200 MHz. Emission was measured at 530 nm with a bandpass filter. For FRET, we used a DNA probe containing a fluorescent donor (fluorescein) at the 5′ end of one strand and an acceptor (rhodamine X) at the 5′ end of the other strand. Labeled DNA concentration was 50 nm. The labeling ratio for acceptor was 99%. The labeling ratio for donor was lower (near 60%), but these unlabeled molecules were invisible when monitoring donor quenching. Bending is detected as enhanced FRET efficiency due to a decrease in end-to-end distance. FRET efficiency was calculated from donor intensity in the absence and presence of acceptor (ID and IDA) as E (E = 1 − (IDA/ID)) and D-A distances (R) were calculated from E using a Ro value for this D-A pair of 55 Å, E = Ro6/(Ro6 + R6) where Ro is the characteristic transfer distance. Fits of the frequency response curves in terms of an energy transfer model with a Gaussian distance distribution were carried out using the Globals Unlimited Program (Laboratory for Fluorescence Dynamics, Urbana, IL). The His-tagged full-length human SRYWT and SRYY129Nproteins were overexpressed in E. coli and purified by nickel chelate affinity chromatography under denaturing conditions, followed by renaturation and size exclusion chromatography as described under “Experimental Procedures.” In comparison with molecular mass standards and other proteins prepared in the laboratory, SRYs eluted as a peak centered at a molecular mass of 22–25 kDa. The molecular mass calculated from the sequence of the gene expressed in E. coli is 25.7 kDa. It therefore appears that full-length SRY is a monomer in solution in the 1–10 μmconcentration range under our purification conditions. The resulting protein is homogeneous by Coomassie Blue staining of a SDS-polyacrylamide gel. Typically, 2 liters of SRY culture yielded 2 ml of a 4 to 8 × 10−6 m solution of pure, native full-length SRY. The target oligonucleotide used was 23 bp in length and derived from the CD3ε gene enhancer, except that it bears the sequence TAACAATG, which allows for 2-fold better binding of SRY HMG box (9Pontiggia A. Rimini R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (255) Google Scholar). Fig. 1 a shows a representative (one of four) anisotropy-based binding isotherm of the 5′-fluorescein-labeled SRY-responsive binding element (F-SRBE) at 2 nm with purified full-length SRYWT at 4 °C in the presence of 50 mm KCl in the TEGD buffer. No change in fluorescence intensity accompanied the increase in anisotropy, and lifetime measurements on the free and bound fluorescein-labeled oligonucleotide revealed an identical 4.2-ns decay. The anisotropy signal thus directly represents a molar quantity. The anisotropy profile for SRY binding to F-SRBE is characterized by an initial plateau for the unbound DNA, followed by a very cooperative anisotropy increase (<1 log unit) as SRY complexes with the fluorescein-labeled DNA and concluded by a long plateau for saturated binding. Under these equilibrium conditions (target [DNA] < C1/2) the apparent midpoint of binding C1/2 was near 12 nm, in agreement with Trimmer et al. (15Trimmer E.E. Zamble D.B. Lippard S.J. Essigmann J.M. Biochemistry. 1998; 37: 352-362Crossref PubMed Scopus (84) Google Scholar). As noted, the anisotropy profile presented a sharp increase in the anisotropy from the initial to saturating plateau between 4 and 30 nmSRYWT, revealing a very large degree of cooperativity. Simple binding of one protein molecule to the DNA would occur over a range of 1.908 log units. Such a high degree of cooperativity rules out simple monomer binding to the target DNA oligonucleotide and demonstrates the existence of an oligomeric SRY-DNA complex. The large shift in the anisotropy values from the initial (95 × 10−3) to the saturating plateau (225 × 10−3), hardly compatible with a monomer binding to DNA, supports this conclusion. We therefore attempted to define the stoichiometry of the complex observed at the saturating plateau. Unlabeled SRBE at a concentration of 45 nm was added to 5 nm labeled F-SRBE (total SRBE, 50 nm) and titrated by SRYWT. At 50 nm SRBE, the oligonucleotide concentration is 5-fold greater than the apparent binding midpoint in Fig. 1 a, thus assuring stoichiometric binding conditions. In the resultant stoichiometric profile (Fig. 1 b), the anisotropy value is observed to continue to increase well beyond a ratio of 1 SRYWT monomer/SRBE molecule, supporting the hypothesis that the complex stoichiometry is not 1:1. In fact, the saturating plateau was reached for a concentration between 295 and 364 nmSRYWT, corresponding to a stoichiometry between 6 and 7 SRYWT molecules/SRBE oligonucleotide. To examine the specificity of the binding, the SRYWT-F-SRBE complex was competed with an excess of unlabeled DNA oligonucleotides (either specific (SRBE) or nonspecific (DR5)). The latter is a 37-bp double-stranded DNA used in the laboratory that bears recognition sequences for the retinoid X receptor-retinoic acid receptor heterodimer and is devoid of any specific binding site for SRY. As observed in Fig. 2 a, an 8- and a 40-fold excess of unlabeled SRBE induced shifts in the titration curves to higher concentrations for the C1/2 (70 and 200 nm, respectively) in the presence of 50 mm KCl. These results (7- and 40-fold displacement of the C1/2 by an 8- and a 40-fold excess of unlabeled specific DNA, respectively) clearly demonstrate the equilibrium status of the interaction between SRYWT and its target DNA under our experimental conditions. Surprisingly, in very similar experiments, a 10-fold excess of unlabeled nonspecific DR5 duplex DNA (Fig. 2 c) resulted in a large shift to a higher concentration of anisotropy increase (C1/2 = 200 nm), revealing a relatively low specificity of the interaction between SRYWT and its target DNA under these low salt conditions. The specificity of the SRYWT binding to F-SRBE was next examined in the fluorescence anisotropy assays by using incremental increases in the buffer salt concentration. Increasing salt concentration usually reduces nonspecific protein-DNA affinity more strongly than specific affinity by competing for interaction with the negatively charged phosphate backbone (18Boyer M. Poujol N. Margeat E. Royer C.A. Nucleic Acids Res. 2000; 28: 2494-2502Crossref PubMed Scopus (65) Google Scholar, 23Record Jr., M.T. Ha J.H. Fisher M.A. Methods Enzymol. 1991; 208: 291-343Crossref PubMed Scopus (276) Google Scholar, 24Ozers M.S. Hill J.J. Ervin K. Wood J.R. Nardulli A.M. Royer C.A. Gorski J. J. Biol. Chem. 1997; 272: 30405-30411Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Increasing salt concentration in the absence of competitor DNA (Fig. 2 b) yielded profiles exhibiting the same saturating plateau, consistent with a complex of identical stoichiometry, but the apparent affinities decreased. At 50 mm KCl, for example, the C1/2 is 12 nm, whereas at 150 mm KCl, it is ∼40 and ∼400 nm at 250 mm KCl. As can be seen in Fig. 2 b, we note a loss of cooperativity at higher salt concentrations that may arise from a salt effect on protein-protein affinity. A striking effect of increasing the salt concentration on the complex specificity can be seen in Fig. 2 c. Closed triangles correspond to the binding of SRYWT to 5 nm F-SRBE in the presence of 250 mm KCl, and the open triangles correspond to the same profile in the presence of 50 nm (a 10-fold molar excess) unlabeled nonspecific target DR5. At this salt concentration, we did not observe the large shift of the binding profile to higher concentration observed with DR5 at 50 mm KCl (closed and open squares). Thus, although SRYWT binds to its target DNA with low specificity at low salt concentrations, presumably due to substantial electrostatic contacts between the positively charged SRYWT and negatively charged DNA, the interaction is of lower overall affinity but becomes much more specific at higher salt concentration. Protein-directed DNA bending is proposed to facilitate the assembly of DNA-multiprotein preinitiation complexes giving rise to architectural gene regulation. Such behavior is believed to be a crucial property of SRY (9Pontiggia A. Rimini R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (255) Google Scholar). Therefore, the complex we observed should bend the target DNA. We have evaluated full-length SRYWT-induced DNA bending by FRET. This technique employs the SRBE probe containing a fluorescent donor (fluorescein) at the 5′ end of the sense strand and an acceptor (rhodamine X) at the 5′ end of the antisense strand. In the absence of protein, for this oligonucleotide, the distance separating the donor-acceptor pair is ∼88 Å. Bending is detected as enhanced FRET efficiency due to decreased end-to-end distance. We have analyzed DNA bending by FRET using increasing amounts of full-length SRYWT, and thus we have established a titration profile of the DNA bending property. Because the saturating anisotropy plateau was identical at all salt concentrations tested, the stoichiometry and nature of the complex are assumed to be similar. We thus evaluated the DNA bending properties of SRYWT at the salt concentration (50 mm KCl) that allowed complete saturation of 50 nm SRBE by SRYWT compatible with our available concentrations of protein. Increasing concentrations of SRYWT ranging from 0 to 800 nm (Fig. 3 a) resulted in a decreased intensity of the donor emission (excited at 450 nm), indicating that FRET occurred upon formation of the SRYWT-SRBE complex. We ascribe this result to protein-induced bending of target DNA. To verify the energy transfer, lifetime measurements on the donor fluorescence were carried out at several concentrations of SRYWT. Binding of SRYWT to the double-labeled F-SRBE-R caused marked changes to the donor fluorescence lifetime (Fig. 4 a). In the free DNA, the mean lifetime of the donor in absence of acceptor (F-SRBE) was 4.2 ns and was unchanged upon protein binding. The presence of the acceptor (F-SRBE-R) did not lead to a reduction in the donor's mean lifetime for the free DNA, consistent with the distance of separation in the linear oligonucleotide. The addition of increasing amounts of SRYWT to the double-labeled target led to complex decay and a progressive shortening of the amplitude-weighted average lifetime (3.25 and 2.44 ns, respectively, at 50 and 100 nm[SRYWT]) before reaching a plateau (0.90 ns for 200 nm [SRYWT]; Fig. 4 b). The fluorescence mean lifetime values were used to calculate FRET efficiency (E) and the distance R between the dyes (Table I). It can be seen from Figs. 3 and 4that for a 1:1 stoichiometry (50 nm both F-SRBE-R and SRYWT), very little FRET occurs, and thus maximal DNA bending was not achieved. Saturation of the FRET signal and thus the protein-induced DNA bending occur near a ratio of 4 SRY/DNA, indicative of the existence of a multiprotein complex on DNA. It is noteworthy that the FRET experiment also revealed DNA bending at elevated salt concentration, as seen in Fig. 3 b, but according to the loss of affinity observed in the presence of 250 mm KCl, the titration was not complete, and the maximal DNA bending could not be achieved at high salt concentration.Table IEnergy transfer parameters for SRY/F-SRBE-RProteinConcentrationR1-aParameters recovered from the analysis of the lifetime data using a model that included the average distance between donor and acceptor and the width of a Gaussian distribution.Width1-aParameters recovered f
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