SRY and Architectural Gene Regulation: The Kinetic Stability of a Bent Protein-DNA Complex Can Regulate Its Transcriptional Potency

Etsuji Ukiyama1,2, Agnes Jancso-Radek2, Biaoru Li, Lukasz Milos, Wei Zhang, Nelson B. Phillips, Nobuyuki Morikawa, Chih-Yen King, Ging Chan, Christopher M. Haqq, James T. Radek, Francis Poulat, Patricia K. Donahoe and Michael A. Weiss

Pediatric Surgical Research Laboratories (E.U., C.M.H., N.M., P.K.D.) Massachusetts General Hospital Harvard Medical School Boston, Massachusetts 02114
Department of Biochemistry (A.J.R., B.L., L.M., N.B.P., C.-Y.K., G.C., J.T.R., M.A.W.) Case Western Reserve University Cleveland, Ohio 44106-4935
Institut de Genetique Humaine (F.P.) UPR Centre Nationale de la Recherche Scientifique 1142 Montpellier 34396, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Protein-directed DNA bending is proposed to regulate assembly of higher-order DNA-multiprotein complexes (enhanceosomes and repressosomes). Because transcriptional initiation is a nonequilibrium process, gene expression may be modulated by the lifetime of such complexes. The human testis-determining factor SRY contains a specific DNA-bending motif, the high-mobility group (HMG) box, and is thus proposed to function as an architectural factor. Here, we test the hypothesis that the kinetic stability of a bent HMG box-DNA complex can in itself modulate transcriptional potency. Our studies employ a cotransfection assay in a mammalian gonadal cell line as a model for SRY-dependent transcriptional activation. Whereas sex-reversal mutations impair SRY-dependent gene expression, an activating substitution is identified that enhances SRY’s potency by 4-fold. The substitution (I13F in the HMG box; fortuitously occurring in chimpanzees) affects the motif’s cantilever side chain, which inserts between base pairs to disrupt base pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA complex to an extent similar to its enhanced function. By contrast, equilibrium properties (specific DNA affinity, specificity, and bending; thermodynamic stability and cellular expression) are essentially unchanged. This correlation between potency and lifetime suggests a mechanism of kinetic control. We propose that a locked DNA bend enables multiple additional rounds of transcriptional initiation per promoter. This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity and decreased lifetime. Aromatic DNA-intercalating agents exhibit analogous kinetic control of transcriptional elongation whereby chemotherapeutic potencies correlate with drug-DNA dissociation rates.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Endocrine systems have long demonstrated the complementary importance of kinetic and equilibrium principles. Gonadotropins, for example, exist in the bloodstream as trapped heterodimers: despite weak binding of {alpha}- and ß-subunits in vitro, dissociation of the functional glycoprotein heterodimer is slow relative to its physiological lifetime (1). Further, insulin analogs of similar affinity for the insulin receptor have been observed to exhibit different mitogenic potency. Although the postreceptor signaling apparatus is incompletely understood, the overall strength of insulin’s signal for DNA synthesis correlates with the lifetime of the hormone-receptor complex rather than its fractional occupancy (2, 3). In this paper we investigate whether the principle of kinetic regulation can extend to the function of an endocrine transcription factor. A model is provided by human SRY, the testis-determining factor encoded by the Y chromosome (4, 5). Our strategy, uncovered in the course of investigating mutations in SRY (6, 7), is similar to that employed in studies of insulin (2, 3). We compare SRY analogs with similar specific DNA affinities but differing kinetics of DNA association and dissociation. Prolonging the lifetime of a bent protein-DNA complex is shown to correlate with enhanced potency in a cotransfection model of SRY-dependent transcriptional activation (6, 8). Our results suggest that the kinetic stability of component DNA bends can regulate the ability of an enhanceosome to support multiple rounds of transcriptional initiation (Fig. 1GoA). To our knowledge, this is the first report of possible kinetic regulation by a specific transcription factor.



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Figure 1. Structure and Proposed Function of SRY as an Architectural Transcription Factor

A, Enhancesome model of architectural gene regulation by SRY. Right, activated transcription occurs in the presence of bound SRY as specific DNA bend permits assembly of stable activator-coactivator-basal preinitiation complex (enhanceosome; Ref. 27). Asterisk (red) indicates site of cantilever insertion. Left, activated transcription is off in the absence of bound SRY due to disassembly of DNA-multiprotein complex and dissociation of activator-coactivator complex. We postulate that the kinetic lifetime of the SRY-DNA complex regulates the overall lifetime of the enhanceosome and thus its ability to support multiple rounds of transcriptional initiation. B, Ribbon model of complex between SRY HMG box (magenta) and 8-bp DNA site (green; Ref. 10). The four hydrophobic wedge side chains (10 ) are also shown in red (top to bottom: W43, F12, I13, and M9). Asterisk indicates site of I13 insertion. C, Ribbon model of SRY HMG showing selected side chains and sites of sex-reversal-associated mutations (red). The residue numbering scheme refers to intact human SRY (41 ) and not the HMG-box consensus (10 ). Asterisks indicate F12 and I13 (F67 and I68 in the intact human sequence). D, Sequences of HMG boxes (HMG-box residues 6–46) highlight conservation of wedge side chains in helix 1 (boxes). First group represents SRY and Sox alleles; middle group, other sequence-specific domains; and bottom group, non-sequence-specific domains. Residue numbers at bottom refer to HMG box consensus (10 ). Conserved wedge side chains are shown in red, and substitutions in blue. E, F12 and I13 (yellow) contact DNA between successive AT bp (5'-ATTGTT; underlined). Coordinates obtained from Ref. 10. F, Crystal structure of TBP (16 17 ). Paired Phe-Phe elements are shown in red and yellow. Whereas the TBP saddle inserts two Phe side chains (one in each lobe as shown in yellow in Fig. 1GoE; Refs. 16 and 17), human SRY employs a single Ile (I13).

 
SRY contains a high-mobility group (HMG) box (9), a conserved motif of minor-groove DNA recognition. The structure of a specific DNA complex exhibits a sharp DNA bend (Fig. 1GoB; Refs. 10, 11, 12). Mutations in the HMG box are associated with 46,XY pure gonadal dysgenesis and sex reversal (sites highlighted in Fig. 1GoC; Refs. 6, 10, 13, 14, 15). The bound DNA structure, in part resembling A-DNA, is similar to that induced by the TATA-binding protein TBP (Fig. 1GoF; Refs. 16, 17). DNA bending is in each case associated with insertion of a nonpolar cantilever side chain between base pairs (Fig. 1GoE). Insertion disrupts base stacking but not base pairing (18). Human SRY contains Ile at the cantilever position, residue 13 of the HMG box consensus sequence. Cantilever side chains otherwise conserved among SRY and related SOX sequences are Phe or Met (Fig. 1GoB). Met occurs in Lef1/Tcf1 HMG boxes (12) where its role is similar to that of I13 in SRY (10). The inserted side chain is in each case flanked by a Phe, which abuts the DNA backbone (Fig. 1GoE). Substitution of either side chain in SRY by Ala abolishes specific DNA-binding activity (19). A mutation in SRY leading to a foreshortened and polar cantilever (I13T) is associated with XY sex reversal and impairs specific DNA binding (6). The variant cantilever accelerates dissociation of the I13T protein-DNA complex by at least 50-fold leading to a proportionate reduction in its association constant. These impaired kinetic and equilibrium properties may each contribute to the failure of testicular differentiation. Analogous partial intercalation occurs among diverse protein-DNA complexes (20, 21, 22, 23, 24, 25, 26). Protein-directed DNA bending is proposed to facilitate assembly of DNA-multiprotein preinitiation complexes [enhanceosomes and repressosomes (27, 28)] giving rise to architectural gene regulation (29, 30).

Experimental distinction between kinetic and equilibrium mechanisms requires identification of amino-acid substitutions that affect one property but not the other. Such substitutions are rare because the two properties are linked: the equilibrium association constant (Ka) is equal to the quotient between rates of association and dissociation (kon/koff). Studies of insulin signaling thus used substitutions that confer compensating changes in rates of receptor binding and release (2, 3). To find such a compensating substitution in SRY, we were guided by an analogy between the HMG box and the chemistry of DNA intercalating agents (31). The latter are organic molecules containing aromatic moieties; examples include daunorubicin, doxorubicin, and adriamycin, agents widely employed in cancer chemotherapy (32, 33, 34, 35, 36, 37, 38, 39, 40). Three considerations informed our structural intuition. First, these drugs insert into DNA to disrupt base stacking but not base pairing, a process reminiscent of insertion of a cantilever side chain (31, 39, 40); second, intercalative agents exhibit a wide range of rate- and association constants, modulated in part by the structure and number of aromatic intercalating moieties (32, 33, 34, 35); third, and most important, transcriptional blockade in vitro and cytotoxicity in vivo correlate with dissociation rates of drug-DNA complexes (36, 37, 38). Kinetic regulation of transcriptional elongation by a DNA-binding agent suggests the possibility of analogous kinetic regulation of transcriptional initiation.

Would an aromatic cantilever in a bent SRY-DNA complex, like that in a chemotherapeutic agent, modulate its kinetic and functional properties? To test this possibility, we exploit a fortuitous experiment of nature: the only difference between the SRY HMG boxes of human beings and chimpanzees is the cantilever substitution I13F (41, 42). Comparison of these HMG boxes demonstrates that the I13F substitution enhances SRY-directed expression of a reporter gene; such expression is absent in control studies of I13T and I13A variants. Despite the I13F variant’s enhanced activity, the substitution appears not to alter the structure, DNA-binding, or DNA-bending properties of the SRY HMG box in accord with a previous study (42). However, 1H-nuclear magnetic resonance (NMR) studies of dissociation kinetics indicate that the lifetime of an F13 complex is prolonged relative to that of an I13 complex. Consistent with studies of transcriptional blockage by DNA intercalative drugs, the extent of kinetic stabilization is similar to the extent of transcriptional enhancement. Accordingly, we suggest that an aromatic cantilever more effectively "locks" the bent SRY-DNA complex in a specific transcriptional preinitiation complex (Fig. 1GoA). This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity but decreased lifetime. Kinetic control of the assembly or disassembly of macromolecular complexes may be a general feature of regulation in diverse biological settings.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional Activation Assay
Because genetic targets of SRY are unknown, we developed a transcriptional assay in a cell line derived from the 14-day rat urogenital ridge [i.e. the waning edge of SRY’s site and stage of expression, 12–14 days post conception (6)]. This assay employs full-length human SRY and uses the Müllerian inhibiting substance (MIS) promoter (43) as a heterologous reporter for transcriptional activation. Cotransfection with a plasmid expressing wild-type SRY leads to a 11-fold induction of transcriptional activity as monitored by a luciferase reporter (Table 1Go; lane k in Fig. 2GoA). Such activation is specific to SRY and its consensus target sequences [5'-ATTGTT-3' (44, 45, 46, 47)] rather than to a structure-specific DNA-binding activity as indicated by the inactivity of human HMG2; further, architectural transcription factors with different DNA sequence specificities [Lef-1 and IRE-ABP with consensus sequences 5'-TTTGAA-3' (12, 44)] are only weakly active in this assay (Fig. 2GoA). Analysis of deletions and base substitutions in the MIS promoter’s HMG-box binding site [5'-TTTGTG; base pairs –48 through –56 relative to the start site of transcription (47)] has shown that activation by SRY persists, presumably through multiple 5'-ATTGTT-3' sites in the vector and reporter.


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Table 1. Effects of Mutations in SRY Structure and Function1

 


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Figure 2. Cotransfection Assay for Transcriptional Activation

A, Fold-induction in luciferase activity is shown relative to a control: lane a, cotransfection of the expression plasmid with an empty vector (1 µg); lanes b and c, 0. 5 and 1.0 µg Sox-2; lanes d and g, 1 µg CDM8; lanes e and f , 0.5 and 1 µg IRE-ABP; lanes h and i, 0.5 and 1 µg Lef-1; and lane j, empty-vector control (1 µg). Ten-fold enhancement is observed after cotransfection with wild-type SRY (lane k; ref.). Error bars represent 2 SD (n = 6). B, Comparison of wild-type, M9I, and I13F SRY variants reveals attenuation of activation by M9I and enhancement of activation by I13F. Error bars indicate 1 SD (n = 6). See also Table 1Go.

 
Although the cotransfection assay does not reflect a physiological effect of SRY on MIS regulation, it nonetheless provides a model of SRY-dependent transcriptional activation. To correlate structure and function, six substitutions in the SRY HMG box were tested (Table 1Go); assays were repeated at least three times to assess statistical significance. Substitutions that reduce specific DNA binding by 10-fold or more in vitro (M9A, F12A, I13A, and I13T) exhibit no detectable transcriptional activation. A substitution that impairs specific DNA binding by 2.5-fold (M9I; Ref. 15) exhibits low but significant activity at high concentrations of vector DNA (1.0–2.0 µg) whereas at lower concentrations any differences from wild type are not significant. Because M9I and I13T are associated with gonadal dysgenesis (10), human phenotypes (male or female) correlate with maintenance or attenuation of transcriptional activity and, in turn, with changes in specific DNA affinity (high or low). Remarkably, the chimpanzee substitution I13F enhances transcriptional activation by 40-fold, which is about 4-fold greater than that stimulated by native SRY (Fig. 2Go). No differences in protein expression were detected in the transfected cells by Western blot using a murine polyclonal antiserum specific for human SRY (Fig. 3GoC). To investigate the origins of the I13F variant’s enhanced activity, native and variant HMG boxes were purified and characterized in vitro. Assays for DNA binding, bending, structure, stability, and kinetics are described in turn below.



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Figure 3. I13F Substitution Does Not Alter Specific DNA Affinity or Base Specificity at Insertion Site

A, GMSA at 4 C. Complex 1 represents specific 1:1 complex with 36-bp probe containing 5'-TGATTGTTCAG target site; complex 2 reflects DNA-dependent protein-protein association. Lanes a–g show binding of I13 SRY domain; lanes i–o show binding of I13F variant. The free DNA is shown in lane h. Successive protein concentrations are as follows: a and i, 2.5 nM; b and j, 5 nM; c and k, 10 nM; d and l, 20 nM; e and l, 40 nM; f and n, 80 nM; and g and o, 160 nM. B, Comparison of variant 15-bp probes containing sequences 5'-ATTGTT, 5'-ATGGTT, 5'-ATAGTT, and 5'-ATCGTT (left panel). GMSA (right panel) indicates that the I13 and F13 domains prefer T at this position, tolerate A, and discriminate to a similar extent against G and C at this position. The protein concentration is 75 nM. "X" indicates faster migrating complex containing a proteolytic contaminant of F13 domain. C, Western blot demonstrates that human SRY (lane 2) and I68F SRY (lane 3) are expressed to similar extents on transfection in CH34 cells (upper panel). Although the antibody may in principle cross-react with rat SRY or other Sox domains, in the Western blot negligible immunoreactive SRY or other proteins less than 40 kDa in molecular mass are detected in an empty-vector transfection control (lane 1). Lower panel shows GH control. In upper and lower panels higher-molecular mass immunoreactive proteins (>40 kDa) document that equal amounts of cell extract were loaded in each lane.

 
DNA-Binding Properties Are Similar
Human and chimpanzee domains exhibit indistinguishable specific DNA binding by gel mobility-shift assay (GMSA) to a 36-base pair (bp) duplex containing a central 5'-ATTGTT-3' consensus site (Fig. 3GoA). In each case a higher-order complex is formed with increasing protein concentration, previously shown to reflect a DNA-dependent protein-protein interaction (48). The two domains exhibit similar GMSA affinities at 4 and 20 C. Sequence specificity was investigated by systematic GMSA shift of variant 15-bp probes (18) containing the four possible base pairs 5' to the insertion site (5'-AXTGTT-3'; X = A, T, C, or G) or 3' to the insertion site (5'-ATXGTT-3'; X = A, T, C or G). These studies (illustrated in Fig. 3GoB for the 3'-X site) demonstrate that the I13F substitution does not alter nucleotide preference at either DNA position. The I13F substitution likewise conferred no detectable change in binding to a four-way DNA junction (data not shown). These results strongly suggest that the enhanced transcriptional activity of the I13F variant is not due to enhanced affinity for the consensus SRY target sequence, variant sequences, or nonspecific prebent DNA.

DNA-Bending Properties Are Similar
Because altered DNA bending may in principle be associated with enhanced or decreased gene-regulatory activity independently of DNA binding affinities (15), SRY-induced DNA bending was measured by permutation gel electrophoresis (PGE) (Ref. 49 ; Fig. 4Go). The DNA site was 5'-CCCATTGTTCTCT-3'. Human and chimpanzee domains exhibit almost identical patterns of position-dependent electrophoretic mobilities. Because interpretation can depend on gel composition, experiments were performed with both 8% and 10% polyacrylamide. Estimated DNA bends in the native complex are 80o and 85o, respectively (inequivalent estimates presumably reflect limitations of the PGE method). Such sharp bending is consistent with prior biochemical studies (45). Binding of human and chimpanzee domains yields similar patterns of flexure-dependent electrophoretic mobilities.



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Figure 4. PGE DNA-Bending Assays Using SRY Target Site 5'-GAGCGCATTGTTATCA Using 8% Gel at 4 C

Lanes 1–6 contain native SRY domain and lanes 7–12 contain I13F variant domain. Experiments were performed using 5'-fluorescein-labeled probes for fluorescent scanning as described in Materials and Methods.

 
Quantitative interpretation of PGE mobilities suggests that I13F substitution implies a small decrement ({Delta}{theta} 2o) in DNA bend angle; this estimate is unaffected by gel composition. Because PGE can be associated with technical artifacts (50), however, SRY-induced DNA bending was also evaluated by phase-modulation fluorescence resonance energy transfer (FRET) (51). This technique employs a DNA probe containing a fluorescent donor (fluorescein) at the 5'-end of one strand and an acceptor (tetramethylrhodamine; TAMRA) at the 5'-end of the other, each tethered by a flexible hexanyl linker. Bending is detected as enhanced FRET efficiency due to a decreased end-to-end distance. The frequency dependence of fluorescence phase and modulation enables donor lifetimes to be obtained in the presence and absence of the acceptor (Table 2GoA). Such lifetimes permit assessment of mean distance changes and modeling of distance distributions (see Discussion).


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Table 2. Analysis of Frequency-Domain Donor Lifetimes (4 C)

 
Binding of the SRY HMG box causes marked changes in phase- and modulation curves (Fig. 5GoA). Similar changes occur on binding of TBP to its DNA site (51). Data may be fitted to a single mean lifetime or with improved fit to two discrete lifetimes (Table 2GoA). In the free DNA the mean lifetime of the donor in the absence of acceptor is 4.44 nsec and is essentially unchanged on protein binding. The presence of the acceptor leads to a reduction in the donor’s mean lifetime to 2.56 nsec in the free DNA. Enhanced FRET on protein binding yields mean lifetimes of 1.79 nsec in the native complex and 1.81 nsec in the I13F complex. The difference ({Delta}{tau} 0.02 ns) is in the same direction as implied by PGE but is within experimental error. Corresponding FRET efficiencies are 0.42 (free DNA) and 0.59 (each complex). Similar FRET efficiencies in the two complexes imply similar mean induced DNA bends and similar distributions of DNA bend angles. Although the extent of FRET enhancement is consistent with a bend angle of 80–85o (51), quantitative interpretation is limited by possible confounding effects of DNA unwinding.



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Figure 5. Phase-Modulation FRET Analysis of DNA Bending

A, Phase- and modulation curves for free DNA (red) and native SRY HMG-box complex (blue). Phase curves are indicated by crosses and modulation curves by circles. B, Gaussian modeling of distance distributions in free DNA (red) and native complex (blue). Lifetimes are given in Table 2GoA and gaussian parameters in Table 2GoB.

 
Structures of Free and Bound Domains Are Similar
A combination of spectroscopic methods [circular dichroism (CD), fluorescence, and 1H-NMR] was employed to test whether the I13F substitution alters structure or stability. The domains exhibit indistinguishable CD spectra in the far ultraviolet, indicating corresponding secondary structures. Intrinsic Trp fluorescence emission spectra likewise exhibit similar quantum yields and emission maxima, probes of tertiary structure. 1H-NMR spectra of free domains are essentially identical. In particular, aromatic and aliphatic spectral regions, which contain sensitive probes for tertiary structure, exhibit corresponding dispersion of chemical shifts and patterns of differential resonance broadening. These residue- specific similarities suggest that the domain’s structure and millisecond dynamics are unaffected by the substitution.

Comparison of two-dimensional (2D) NMR spectra reveals the new F13 aromatic spin system, which exhibit properties expected of an unhindered side chain at a protein surface: negligible secondary shifts, motional narrowing, and absence of nonlocal nuclear Overhauser effects (NOEs). Specific DNA complexes exhibit a similar correspondence of NMR spectra (Fig. 6Go). The upfield methyl resonances of V5 and L46 provide probes for the DNA-bound structure of the minor and major wings, respectively (10). As expected, the unusual aliphatic cantilever resonances of I13 [shifted upfield by the ring currents of flanking DNA base pairs (18)] are absent in the variant complex. Together, the spectroscopic results suggest that the enhanced activity of the I13F protein is not due to a nonlocal change in the structure of the free or bound HMG box.



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Figure 6. I13 and F13 SRY-DNA Complexes Exhibit Similar 1D 1H-NMR Spectra at 500 MHz and 37 C

Upfield aliphatic spectra regions of native complex (A) and I13F variant complex (B). Native and variant complexes exhibit similar upfield shifts of the methyl resonances of V5 (induced fit of minor wing; Refs. 68 and 69) and L46 (major wing). L46 is also shifted upfield in free domains, indicating preformed structure of major wing. Asterisk indicates anomalous upfield ß methylene resonance of L46 due to ring current of W43 in core. I13 in native complex is shifted far upfield due to ring currents of flanking base pairs; this spin system is absent in spectrum of I13F variant.

 
Stabilities of Free and Bound Domains Are Similar
Thermodynamic stabilities of native and I13F domains were investigated by titration with denaturant guanidine hydrochloride. The extent of unfolding was monitored by Trp fluorescence. The two domains exhibit similar thermodynamic parameters as inferred from a two-state model of an equilibrium between native and unfolded states (Table 3Go). Respective concentrations of guanidine at which 50% of the domain is unfolded (Cmid; column 2 of Table 3Go) are essentially identical. A slight difference in free energies ({Delta}G{upsilon}; column 4) is suggested, but inferred differences are within experimental error ({Delta}{Delta}G{upsilon} 0.25 ± 0.26 kcal/mol). Thermal stabilities, evaluated by measuring the temperature dependence of both CD ellipticity (at helix-sensitive wavelength 222 nm) and Trp fluorescence, are also similar (unfolding midpoints 40 ± 1 C and 42 ± 1 C, respectively; Table 1Go). Because of the breadth of the unfolding transition in the range 30–50 C (47), fractional unfolding at physiological temperature (37 C) would not differ significantly. These in vitro results are in accord with the similar biological expression stabilities of native and I13F variant SRY in cell culture (see above).


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Table 3. Thermodynamic stabilities of SRY HMG Box

 
I13 and F13 Complexes Exhibit Different Kinetic Lifetimes
NMR spectroscopy enables kinetic lifetimes of native and I13F variant complexes to be estimated. The assay exploits the observation that resolved DNA imino resonances exhibit large complexation shifts on drug- or protein binding. Spectral changes can be slow, intermediate, or fast on the time scale of 1H-NMR chemical shifts (33, 34).

Imino resonances of a 15-bp DNA duplex containing a central 5'-ATTGTT-3' target site exhibit large changes in chemical shift on specific binding of the SRY domain. Titration with the I13F domain (spectra c–e in Fig. 7GoA) reveals that free and bound DNA are in slow exchange on the 1H-NMR chemical-shift time scale as previously described in the native complex (18). Arrows in Fig. 7GoA–c indicate discrete DNA imino resonances from a specific complex in equilibrium with free DNA. Prominent is the anomalous position of the T8 imino resonance, which is upfield among the guanine imino resonances (Fig. 7GoA–e). I13 and F13 complexes exhibit significant differences in imino 1H-NMR signatures (Fig. 7Go). These differences are consistent with the ring current of an aromatic cantilever: i.e. upfield differences are observed at flanking thymidine imino and adenine H2 positions (hence above and below the F13 aromatic ring), whereas smaller downfield differences occur at the edge of the insertion site flanking (side-chain protons of F12 and W43). This pattern implies planar (rather than perpendicular) insertion of F13 between base pairs, consistent with the shape of the cavity induced in the DNA and analogous to planar intercalative agents (34, 39, 40). The slow-exchange condition provides a lower bound on the lifetime of the bound state: slow exchange between resonances separated by 0.1 ppm (40 Hz at a 1H-NMR resonance frequency of 400 MHz) implies a lifetime greater than 25 msec.



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Figure 7. DNA Imino Resonances and Kinetics of Protein-DNA Dissociation

A, 1H-NMR titration of 15-bp DNA duplex by I13F domain at 400 MHz; a and b, spectra of free DNA and native SRY-DNA complex; c and d, addition of successive aliquots of I13F domain leads to appearance of new set of imino resonances (arrows in c) in slow exchange; and d, spectrum of 1:1 I13F variant complex. Assignments are as indicated. The three indole NH resonances of tryptophan are as labeled between 10 and 11 ppm. Asterisks in panel e indicate insertion site of cantilever side chain (AT base pairs 8 and 9); asterisk in panel b indicates perturbation in W43 at protein-DNA interface. Imino resonances of base pairs 1 and 15 are not seen due to fraying; the resonances of base pairs 2 and 14 are broadened. B and C, Chemical-exchange cross-peaks of equimolar mixtures of free and bound DNA at 40 C at 500 MHz. A representative trace is shown in each case to illustrate the ratio of diagonal resonance intensity to cross-peak intensity; asterisks indicate cross-peak. Smaller resonances in trace in panel B indicate neighboring base pairs 11 and 9 in the bound state. Assignments of exchange cross-peaks are as indicated. The DNA concentration in (A) was 2 mM in panel A and in (B) was 0.5 mM in panel B.

 
Slow exchange gives rise to cross-peaks in 2D NOESY spectra of equimolar mixtures of free and bound DNA. Cross-peaks (obtained with a mixing time of 200 msec as shown in Fig. 7Go, B and C, for native and variant complexes) connect the diagonal resonances of a given DNA proton in the free and bound states. Representative traces (inset in Fig. 7Go, B and C) enable comparison of the relative extent of exchange. Whereas in the wild-type complex the cross-peak intensities are {approx} 65% of those of diagonal resonances, in the variant complex their intensities are significantly lower ({approx}15–20%; asterisks in Fig. 7Go, B and C). Native exchange cross-peaks are consistent with a lifetime similar to the NOESY mixing time whereas the lifetime of the chimpanzee complex is prolonged 3- to 4-fold. Gel-shift studies of the 33P-labeled 15-bp duplex in NMR buffer indicate similar apparent binding of the two domains at 4, 20, and 37 C. In each case apparent binding is substantially weaker than that observed using a 36-bp probe (see Fig. 3Go, A and B). At 37 C and 100 nM protein concentrations, for example, the percent shift is only 4 ± 1%. It is likely that the intensity of the shifted bands is attenuated by rapid dissociation of native and variant complexes on the time scale of electrophoresis. A dissociation constant is the ratio of the rates of association and dissociation. Because apparent affinities are similar, the prolonged lifetime of the I13F domain indicates a similarly prolonged association rate. Comparison with kinetic studies of a non-sequence-specific HMG box [HMG1-A recognition of a cisplatin-DNA adduct (52)] implies that sequence-specific DNA binding is significantly slower (both in association and dissociation) than structure-specific DNA binding.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA-bending proteins containing an HMG box have attracted broad interest due to their roles in mammalian organogenesis (53, 54, 55). Because these proteins may lack discrete domains of transcriptional activation or repression, such architectural transcription factors are proposed to regulate gene expression in part through changes in the structure of DNA or chromatin (29, 30). A model in which introduction of a specific DNA bend enables cooperative assembly of a DNA-multiprotein preinitiation complex is shown in schematic form in Fig. 1GoA. In this article we have compared SRY analogs to assess a proposed chemical and functional analogy between the HMG box and aromatic DNA-binding drugs (31). On intercalative DNA binding, such drugs disrupt base stacking but seldom base pairing and often induce features of A-DNA. Our strategy exploits a fortuitous experiment of nature, the aromatic cantilever of chimpanzee SRY (F13; Refs. 41, 42). Although the equilibrium properties of I13 and F13 domains are similar, an aromatic cantilever enhances potency in a model transcriptional assay in association with enhanced kinetic stability.

Phase-Modulation FRET Assay Implies Well Defined Bend Angle
Electrophoretic anomalies have been widely used as probes of DNA bending. Interpretation of PGE may be confounded, however, by geometric features of a protein-DNA complex (50). As a control for such artifacts, we have employed a complementary FRET assay. FRET efficiency reflects the distance between 5'-ends of the DNA, which is a function of both DNA bending and DNA unwinding. A similar assay has been described in studies of TBP (51). The present studies are in accord with this and previous PGE studies of HMG-box complexes (15, 42, 45). Electrophoretic- and FRET methods both indicate that the I13F substitution has at most a small effect on induced DNA bending; a decrement of {Delta}{theta} 2o as estimated by PGE is within the error of the FRET measurements. Assuming no change in orientation factor {kappa}2, corresponding mean FRET distances are 58.9 Å ± 1 Å in the free DNA (using a measured Ro value of 55.9 Å) and 51.1 Å ± 1 Å in the complexes (using a measured Ro value of 54.3 Å). The reduction in mean distance is hence about 8 ± 2 Å, which is consistent with a bend angle of 80–85o. The single mean-lifetime and single mean-distance approximations neglect the actual distribution of distances in solution that arise from motions of the linkers, dyes, and DNA. These approximations are nonetheless of qualitative value and have been successfully applied in FRET studies of HMG-1a (52). Although the relationship between end-to-end distance and bend angle is not straightforward (due to variable DNA unwinding), the FRET observations corroborate PGE results to demonstrate that the DNA-bending properties of the two proteins are similar and so unlikely to be the source of the variant’s enhanced transcriptional activity.

Neither PGE nor single-lifetime FRET analysis distinguishes between static and dynamic changes in DNA bending. A mutation in a protein may change the mean DNA bend angle, for example, without change in fluctuations. Alternatively, a mutation may induce dynamic instability or imprecision in DNA bending, leading to a larger distribution of populated angles. By enabling an estimate of distance distributions, FRET techniques can, in principle, provide insight into this distinction (56). Thus, the present frequency-domain data of donor-acceptor samples are much better fit by two discrete lifetimes than by a single lifetime (Table 2GoA). These lifetimes can be interpreted according to a gaussian distribution of distances (51, 56). This analysis, which also assumes that isotropic orientation factor {kappa}2 equals 2/3, in each case, suggests that binding of the I13 and F13 domains induces a 9 Å ± 1 Å decrease in end-to-end distance (difference between <r> values for free and bound DNA) with no significant change in widths of the distributions (Fig. 5GoB and Table 2GoB). The widths are due predominantly to reorientation of the linkers. These results are comparable to two prior FRET studies of DNA bending induced by nonspecific HMG boxes (52, 57).

In the absence of an adequate cantilever side chain, would a variant SRY-DNA complex be sharply bent and if so, would significant fluctuations occur in bend angle? In the future this question may be addressed through studies of additional analogs. It would be of interest to find a variant cantilever sufficient to provide near-native specific binding affinity (or equivalently, free energy {Delta}G) but associated with nonlocal dynamic instability. The plausible existence of such analogs arises from consideration of enthalpy-entropy compensation (58): reduced enthalpy of side-chain insertion ({Delta}H) may be compensated by the enhanced entropy of a fluctuating complex ({Delta}S). Such analogs would enable the regulatory importance of precise and stable DNA bending to be tested. A signature of such a variant complex would be an anomalously broad distribution of end-to-end distances.

Mutation Enhances Transcriptional Potency and Kinetic Stability
The present study extends previous studies of SRY (13, 14, 15, 42) by correlating phenotypes and biochemical activities with transcriptional regulation. Although the mechanism of SRY-dependent transcriptional activation in our assay is unknown and not specific to MIS, our vector-reporter system nonetheless provides a model of ATTGTT-dependent transcriptional activation. Our study has compared functional proteins (human SRY and I13F chimpanzee variant) and nonfunctional proteins (clinical variants M9I and I13T and control mutations F12A and I13A). Whereas residues I13 and F13 are each compatible with the male phenotype in a primate, a polar and foreshortened cantilever (I13T) or altered hydrophobic wedge (M9I) blocks testicular differentiation (6, 10, 13, 14, 15). These and other substitutions with decreased or undetectable specific DNA binding exhibit decreased or undetectable SRY-dependent transcriptional activation (Table 2Go).

Because the chimpanzee SRY HMG box exhibits native DNA affinity, bending, and nucleotide specificity at the insertion site, we hypothesize that the variant’s enhanced activity in the cotransfection assay is due to enhanced kinetic stability: the bent protein-DNA complex is more stably maintained once formed in a preinitiation complex. Physical evidence for slower dissociation (relative to the native complex) is provided by 1H-NMR. Quantitative implications for the kinetics of protein binding and release may be estimated as follows. The change in barrier height corresponding to a factor of 4 in on- or off rates ({Delta}{Delta}G+) would be modest (0.8 kcal/mol). A lifetime of 200 msec at 40 C (consistent with the native NMR data) would imply a dissociation rate of 5 sec-1. Assuming an equilibrium constant of 50 nM under these conditions, this would imply an association rate of 108 M-1sec-1. This is 10-fold slower than the rate of association of HMG1 to a prebent DNA cisplatin adduct at 25 C (koff 30 sec-1 and kon 109 M-1sec-1; Ref. 52). We propose that the rate-determining step in SRY binding occurs in the pathway of HMG box-directed DNA reorganization and that the protein cantilever participates in this step. The analogous kinetic barrier in the adducted HMG1 domain complex is presumably reduced by distortion of the free platinated DNA. Stopped-flow kinetic studies of the binding of HMG-1a to cisplatin-modified DNA has recently shown that although the association rate is near the diffusion limit, significant differences in association rates (but not dissociation rates) are observed when the DNA sequences are varied at the site of platination (59). The present study, by contrast, highlights a difference in dissociation rates. The slower dissociation rate for specific SRY-DNA complexes is consistent with studies of lymphoid enhancer factor 1 (LEF-1) ( by competition kinetic GMSA (estimated lifetime 120 min or koff 1.1 x 10-4 sec-1 at 20 C; 60). Surface plasmon resonance studies of HMG1 and HMG2 (which each contain two HMG box domains) indicate that bidentate binding to a 30-bp duplex exhibits kinetic constants of 2 x 104 M-1 sec-1 (kon) and 6–7 x 10-2 sec–1 (koff) (61).

Intriguingly, the prolongation of I13F variant’s lifetime is similar to its extent of enhanced transcriptional activation. Although this could be a coincidence, the absence of a plausible alternative mechanism suggests a model of kinetic regulation in which the net accumulation of mRNA is controlled not by the equilibrium constant of the SRY-DNA complex but by its rate of dissociation from an activated preinitiation complex. This model further posits that, of the many protein-DNA interactions in a preinitiation complex, the lifetime of the SRY-DNA complex can be the limiting parameter. We imagine that once SRY dissociates and the DNA unbends, multiple other protein-DNA and protein-protein interactions are weakened (Fig. 1GoA). Accordingly, we propose that kinetic stability of the bent SRY-DNA complex enables maintenance of the preinitiation complex through multiple rounds of transcription. Although the physiological significance of this observation is not clear (as the present cotransfection assay does not use a bona fide SRY target promoter and, in any case, both I13 and F13 ultimately results in male differentiation in the appropriate primate), it is possible that SRY-responsive genes in vivo will be found to exhibit diverse promoter/enhancer structures with distinct rate-limiting steps in respective pathways of preinitiation complex assembly and disassembly.

We caution that the present data are only correlative. Confounding effects in the intracellular milieu of our gonadal cotransfection assay (such as differential nuclear targeting, and differential binding to bystander proteins in the cell) cannot be excluded. Because the substitutions at position 13 are distant from possible phosphorylation sites and nuclear localization signals, however, such effects seem unlikely. It is also possible that the I13 and F13 domains have different affinities for a 5'-ATTGTT-3' site within a nucleosome, a structural target not tested in the present study. Despite these caveats, a kinetic constraint on SRY activity would be broadly consistent with kinetic control in other nonequilibrium systems as discussed below.

Is Kinetic Control a General Biological Phenomenon?
The signature of kinetic control is a correlation between rates (rather than equilibrium constants) and a functional endpoint. Examples in endocrinology include the kinetic trapping of gonadotropin heterodimers (1) and kinetic control of mitogenic signaling by insulin (2, 3). Kinetic control has also been established among serpin proteases and has physiological relevance (62). The misfolding of proteins and subsequent trapping of non-ground-state forms within aggregates are proposed as pathogenic steps in prion-related encephalopathies (63). Kinetic control of a processive enzymatic process may likewise be effected by rates as exemplified by transcriptional elongation: rapidly dissociating drug-DNA complexes do not hinder elongation even if compensated by rapid association rates leading to similar equilibrium constants. Model studies have used T7 RNA polymerase (a small single-subunit polymerase) and the multisubunit bacterial RNA polymerase of Escherichia coli (36, 37, 38). Slowly dissociating intercalative agents cause prolonged pausing of the polymerase preceding the site of intercalation, in turn enhancing the probability of transcriptional termination with release of the enzyme from the transcription bubble. The faster and less stable T7 enzyme terminates at each slowly dissociating ligand complex. Actinomycin D, echinomycin, mithracycin, and noglamycin provide examples of slowly dissociating drugs (t1/2 > 5 min) whereas adriamycin provides an example of a rapidly dissociating drug (t1/2 < 1 sec). A metaphor of a train (the processive RNA polymerase) being derailed by a car on the tracks (the intercalative complex) is supported by the correlation between diminished bioactivity and creep rate or lateral diffusion of the drug along the DNA. Enhanced lateral diffusion by an intercalative agent (visualized as a car outracing the train) correlates with impaired bioactivity even if the overall rate of drug-DNA dissociation is slow (35).

Summary
Structure-function relationships in the SRY HMG box have been inferred from comparative studies of clinical variants (6, 10, 13, 14, 15, 44, 45). By correlating phenotype with biochemical activities, such studies have suggested that SRY’s genetic function requires specific DNA binding (44, 45) and DNA bending (15). The present study has focused on comparison of primate SRY HMG boxes (42), which differ only by the presence of an aliphatic- (human, gorilla, orangutan, baboon, and marmoset) or aromatic (common and pygmy chimpanzees) cantilever. This comparison was motivated by an analogy proposed between DNA-bending proteins and intercalative DNA-binding drugs (31). The results highlight the importance of the cantilever mechanism and suggest a new avenue of investigation, kinetic control of transcription. In the future it would be of interest to extend this analysis to M13 (the most common cantilever among mammalian Sry and SOX sequences; Fig. 1GoD). Characterization of this and diverse side chains of chemical interest may further test the proposed relationship between kinetic stability and transcriptional activation.

Future dissection of the structural, kinetic, and thermodynamic factors underlying SRY-dependent gene regulation will require identification of physiological target genes and biochemical reconstitution of sex- and tissue-specific preinitiation complexes. It is likely that variant HMG boxes with well defined biochemical properties will provide valuable tools to uncover target genes and explore mechanisms of regulation. Our hypothesis of kinetic control predicts the occurrence of novel clinical mutations in HMG boxes that accelerate rates of specific DNA association and dissociation but do not impair DNA affinity. The SRY system may provide an opportunity to investigate a nonequilibrium mechanism of transcriptional regulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Protein Purification
The SRY HMG box (85 residues) was expressed in E. coli as a thrombin-cleavable fusion protein as described (18). Final purification was accomplished by FPLC using a MonoS column (Pharmacia Biotech, Piscataway, NJ). HMG box fragments were obtained both with and without C-terminal His6 tag. The tag does not affect protein folding (as determined by CD and 1H-NMR) or specific DNA binding (as determined by GMSA). Purity was more than 98% as assessed by SDS-PAGE.

Site-Directed Mutagenesis
Substitutions were introduced in phage M13 mp19RF by oligonucleotide-directed mutagenesis (6) and recloned by PCR into an expression plasmid (18). Constructions were verified by DNA sequencing.

Four-Way DNA Junctions
Four 30-bp oligonucleotides J1 were prepared as described (7). One strand was labeled with 32P, annealed overnight with the other three strands from 80 C to 4 C in 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 10 mM MgCl2, and 50 mg/ml BSA. Efficiency of junction formation was more than 80% as estimated by 12% PAGE.

Cell Culture and Western Blot
Cell line CH34 (6) was cultured in DMEM containing 5% heat-inactivated FBS and 1x penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD) at 37 C under 5% CO2. Cotransfections were performed using the lipofectAMINE 2000 kit (Life Technologies, Inc.). Briefly, after cells were washed twice with 1x PBS, SRY expression plasmids were cotransfected with pXGH5 (6) in 1:1 ratio as an internal control for transfection efficiency and expression level. After overnight cotransfection, fresh medium was added. For Western blots, identical aliquots of cell extracts were electrophoresed on 10% SDS-PAGE. The gel was blotted on nitrocellulose membrane (Bio-Rad Laboratories, Inc. Hercules, CA). Hybridization solutions containing anti-HMG domain antibody SE75 at a concentration of 1:100 or antihuman-GH antibody (obtained from Sigma, St. Louis, MO) at a concentration of 1:100, respectively, were hybridized onto the membrane. After goat antirabbit IgG was incubated for 1 h, a chemiluminescent detection system (Bio-Rad Laboratories, Inc.) was used for specific protein detection. Antibody SE75 is specific for the HMG box of human SRY and was raised as described (64). Cross-reactivity to SOX HMG box of a 67 kDa can occur (P. Berta, personal communication). The present Western blots contained no bands less than 40 kDa in the control transfection (thus excluding detection of rat Sry) and only a single band of size appropriate to human SRY in the SRY- and I68F-SRY-transfected lanes.

DNA-Binding Assay
Oligonucleotides were purchased from Oligos, Etc. (Wilsonville, OR). 1) The duplex probe was labeled with 32P or 33P, annealed, and analyzed using the gel-retardation assay (18). Each reaction contained 2.5–160 nM protein (see caption to Fig. 4GoA) and less than 1 nM labeled DNA in 10 mM potassium phosphate (pH 7.0), 50 ng/ml BSA, 50 mM KCl, 4 mM dithiothreitol, and 2.5 mM MgCl2; the reaction was incubated for 1 h on ice. Only specific binding is observed under these conditions. 2) For analysis of four-way DNA junctions, reactions contained 25–250 nM protein and 4 nM labeled junction in the same buffer. The 36-bp probe contains the DNA sequence 5'-CATACTGCGGGGGTGATTGTTCAGGATCATACTGCG-3' and complement (target site underlined). The parent 15-bp probe (also used for NMR studies) contains the sequence 5'-GTGATTGTTCAG-3' and complement. To assess sequence specificity, variant duplexes 5'-GTGAXTGTTCAG-3' and 5'-GTGATXGTTCAG-3' (X = other base pairs) with respective complements were similarly prepared.

Electrophoretic DNA-Bending Assay
Double-stranded oligonucleotides containing consensus SRY binding site 5'-CCCATTGTTCTCT-3' and complement were cloned between XbaI and SalI sites of vector pBend2 (65). Probes of equal length (147 bp) with the binding site at varying distance from ends (distances of bend center from 5'-end are 120, 95, 79, 51, 47, and 27 bp) were generated by PCR, and 5'-labeled with 33P-ATP using T4 polynucleotide kinase. Ten microliter binding reactions contained 50 mM KCl, 20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 50 ng poly(dI-dC), 10% glycerol, approximately 1 nM 33P-labeled DNA probe, and 60 nM SRY-HMG box domain. After 1–2 h incubation on ice, samples were run on polyacrylamide gels in 0.5x (0.045 M) Tris-borate buffer containing EDTA (TBE) at {approx} 10 V/cm.

Steady-State Tryptophan Fluorescence and Stability Measurements
Steady-state fluorescence spectra and automated guanidine titrations were obtained using an Aviv spectrofluorimeter (model ATF105, Aviv Instruments, Lakewood, NJ). Intrinsic Trp fluorescence was measured at an emission frequency of 390 nm (slit width 5 nm) after excitation at 270 nm (slit width 2 nm) at 4 C. Denaturation studies of the human domain as monitored by CD at 222 nm yielded similar results. Human and chimpanzee SRY domains were made 1 µM in 140 mM KCl, 10 mM potassium phosphate (pH 7.4) in the titrating cuvette. The same concentration of SRY was used in the titrant reservoir containing 7.2 M guanidine-HCl in the above buffer. Experimental curves were fitted by nonlinear least squares to the equations: {Delta}G = {Delta}Gu + m x [guanidine] = –RT x ln K, where {Delta}Gu is the free energy of unfolding extrapolated to zero denaturant concentration and K is the equilibrium constant between native and unfolded states of the protein (66).

Phase-Modulation Fluorescence and FRET
Time-resolved measurement were performed on a SPEX Fluorolog {tau}-2 Lifetime Spectrofluorometer (Hoboken, NJ). The light source was a xenon lamp with excitation wavelength of 490 nm. The frequency of the excitation beam was modulated in a Pockel cell between 20 and 200 MHz. Sample response was monitored at the emission wavelength of fluorescein (518 nm) by a photomultiplier (PMT) through a Corion 520 band-pass filter (ThermoCorion Inc., Franklin, MA) at magic angle. Time-resolved measurement were performed on 15 µM samples. The reference was LUDOX TM-50 colloidal silica suspension (DuPont Merck Pharmaceutical Co., Wilmington, DE) obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Measurements were performed at 4 C. Fitting analysis, performed on Globals Unlimited software (Laboratory for Fluorescence Dynamics, University of Illinois, Urbana-Champaign, IL), indicated that fluorescein’s spectral response could be best described with double-exponential curve with two distinct lifetimes contributing to phase-response (51). Fitting parameter space was changed to target parameters representing physical invariant of system, after which a complete system could be specified. For fluorescein-rhodamine pair energy transfer was assumed to give rise to two lifetimes. The Förster distances for free and bound DNA were measured to be 56 Å and 54 Å, respectively, and the distance between chromophores was assumed to vary according to normal distribution. Parameter optimization was performed to minimize overall {chi}2. Error levels of 0.2 (phase) and 0.01 (modulation) are assumed for analysis. FRET measurements were obtained in 50 mM KCl and 50 mM Tris-borate (pH 7.4).

CD Spectroscopy
Spectra were obtained using an Aviv spectropolarimeter equipped with thermister temperature control for automated analysis of thermal melting. Samples were observed in a 1-mm pathlength quartz cuvette in a buffer consisting of 10 mM potassium phosphate (pH 7.4) and 50 mM KCl.

NMR Spectroscopy
Spectra were observed at 400 and 500 MHz using Varian spectrometers (Varian Instruments Inc., Palo Alto, CA) at Harvard Medical School and The University of Chicago. Spectra of exchangeable resonances were obtained in H2O using laminar-shifted shaped pulses in the absence of solvent presaturation as described (18). Spectra of free domains were obtained at 25 C in 10 mM deuterated acetic acid (pH 4.5) and 140 mM KCl; such spectra are similar in the pH range 4–8. Spectra of complexes were obtained at 40 C in 10 mM potassium phosphate (pH 6.0) and 50 mM KCl.


    ACKNOWLEDGMENTS
 
Acknowledgements

We thank E. Haas and V. Itah (Bar Ilan University, Ramat Gan, Israel) for measurements of fluorescent Ro values and advice regarding FRET methods; W. Olson and A. R. Srinivasan (Rutgers University, Newark, NJ) for discussion of FRET design and linker dynamics; P. Berta (Institut de Genetique Humaine, Montpellier, France) for anti-SRY antisera; D. Love and P. E. Wright (Scripps Research Institute, La Jolla, CA) for CURVES analysis of Lef1-DNA and SRY-DNA structures; W. Jia for assistance with NMR measurements and processing; G. L. Waneck for advice regarding site-directed mutagenesis; and L. Labeots and M. Mellody for assistance with DNA binding studies.


    FOOTNOTES
 
Address requests for reprints to: Michael A. Weiss, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935. E-mail: maw21{at}po.cwru.edu or Patricia K. Donahoe, Pediatric

This work was supported in part by grants from the NIH to P.K.D. (Grant CA-17393) and M.A.W. (Grant CA-63485).

1 Present Address: Urawa Municipal Hospital, Department of Pediatric Surgery, 2460 Mimuro Yrawa-shi, Saitama 336-8522 Japan. Back

2 These authors contributed equally. Back

Received for publication September 13, 2000. Revision received January 2, 2001. Accepted for publication January 4, 2001.


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