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DNA Bending Is Essential for the Site-specific Recognition of DNA Response Elements by the DNA Binding Domain of the Tumor Suppressor Protein p53

1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês

10.1074/jbc.272.23.14842

1083-351X

Akhilesh K. Nagaich, Ettore Appella, Rodney E. Harrington,

Molecular Biology Techniques and Applications

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is ∼50°), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from ∼37 to ∼25°). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function. We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is ∼50°), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from ∼37 to ∼25°). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function. The wild type tumor suppressor protein p53 plays a critical role in many key cellular processes (1Lane D.P. Nature. 1992; 358: 15-16Google Scholar, 2Levine A.J. Annu. Rev. Biochem. 1993; 62: 623-651Google Scholar, 3Ko L.J. Prives C. Genes & Dev. 1996; 10: 1054-1072Google Scholar). It acts as a transcriptional activator for a number of DNA damage and growth arrest genes includingmdm2, gadd45, and p21/waf1/cip1; the product of the last is directly involved in inhibiting Cdk complexes leading to cell cycle arrest at the G1/S phase checkpoint (4Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Google Scholar, 5Kastan M.B. Zhan Q. El-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Google Scholar, 6El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Google Scholar, 7Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Google Scholar). In addition to this protective role, p53 also up-regulates the human bax gene, the product of which heterodimerizes with the survival factor Bcl-2 and directly controls the apoptotic process (8Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Google Scholar). p53 also negatively regulates the transcription of genes that have TATA box-initiated promoters by binding to the protein components of the basal transcription machinery and is thought to be directly involved in checking both viral and eukaryotic DNA replication (9Horikoshi N. Usheva A. Chen J.D. Levine A.J. Weinmann R. Shenk T. Mol. Cell. Biol. 1995; 15: 227-234Google Scholar, 10Bargonetti J. Friedman P.N. Kern S.E. Vogelstein B. Prives C. Cell. 1991; 65: 1083-1091Google Scholar, 11Dutta A. Ruppert S.M. Aster J.C. Winchester E. Nature. 1993; 365: 79-82Google Scholar). Wild type p53 binds response elements through a sequence-specific DNA binding domain (p53DBD) 1The abbreviations used are: p53DBDp53 DNA binding domainbpbase pair(s)RGCribosomal gene clusterMES4-morpholineethanesulfonic acidbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol extending from amino acid residues 96–308 (12Bargonetti J. Manfredi J.J. Chen X. Marshak D.R. Prives C. Genes & Dev. 1993; 7: 2565-2574Google Scholar). Studies of tumor-derived p53 mutants have shown that they are defective in sequence-specific DNA binding and consequently cannot activate transcription (13Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Google Scholar). These studies strongly suggest that sequence-specific DNA binding and transactivation are the key biochemical activities responsible for much of the biological function of p53. Mutations in the p53 protein have been associated with more than half of all forms of human cancers, and the p53gene is thought to be the most frequently mutated gene in human cancer (13Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Google Scholar). Over 1000 tumor-derived mutations have been found in p53, and the vast majority of these mutations are located in the p53DBD and affect its sequence-specific DNA binding (14Harris C.C. Science. 1993; 262: 1980-1981Google Scholar). This fact, supported by many biochemical and molecular genetic experiments, has suggested that p53 function is mediated by its DNA binding activity and transactivation properties (12Bargonetti J. Manfredi J.J. Chen X. Marshak D.R. Prives C. Genes & Dev. 1993; 7: 2565-2574Google Scholar, 15Pavletich N.P. Chambers K.A. Pabo C.O. Genes & Dev. 1993; 7: 2556-2564Google Scholar). p53 DNA binding domain base pair(s) ribosomal gene cluster 4-morpholineethanesulfonic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol Wild type p53 binds over 100 different naturally occurring response elements associated with different specific functions; it has been estimated that the human genome may contain as many as 200–300 such sites (16Tokino T. Thiagalingam S. El-Deiry W.S. Waldman T. Kinzler K.W. Vogelstein B. Hum. Mol. Genet. 1994; 3: 1537-1542Google Scholar). p53 response elements differ in details of specific base sequence, but all contain two tandem decameric elements or half-sites, each a pentameric inverted repeat. Most decamers follow the consensus sequence pattern RRRC(A/T)‖(A/T)GYYY, where R and Y are purines and pyrimidines, respectively, and the vertical bar denotes the center of pseudodyad symmetry (17El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Google Scholar). One copy of these decameric half-sites is insufficient for the functional binding of p53, defined as the ability to transcriptionally activate a nearby reporter gene, but some binding is preserved when the two copies are separated by up to 21 bp (16Tokino T. Thiagalingam S. El-Deiry W.S. Waldman T. Kinzler K.W. Vogelstein B. Hum. Mol. Genet. 1994; 3: 1537-1542Google Scholar). A cocrystal structure of the p53DBD nucleoprotein complex (18Cho Y.J. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Google Scholar) has provided valuable insights into the binding specificity of p53 by identifying specific binding contacts (3Ko L.J. Prives C. Genes & Dev. 1996; 10: 1054-1072Google Scholar, 19Prives C. Cell. 1994; 78: 543-546Google Scholar, 20Prives C. Bargonetti J. Farmer G. Ferrari E. Friedlander P. Wang Y. Jayaraman L. Pavletich N. Hubscher U. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 207-213Google Scholar). However, since the asymmetric unit contained only a single normally bound p53DBD, many questions remain concerning the multisubunit nature of the full p53 nucleoprotein complex, the determinants of DNA binding specificity, and the overall organization of p53 tetramers bound to the DNA recognition site. A recent study using T4 ligase-mediated cyclization and analytical ultracentrifugation has shown that p53DBD binds cooperatively as a tetrapeptide to an important functional response element, p21/waf1/cip1, and induces substantial bending in the DNA (21Balagurumoorthy P. Sakamoto H. Lewis M.S. Zambrano N. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8591-8595Google Scholar). More recent studies from this laboratory have shown that the requirement of DNA bending is maintained in dipeptide p53DBD complexes with several half-sites, which form at lower binding affinities than tetrapeptide complexes with full response elements. 2P. Balagurumoorthy, unpublished results. 2P. Balagurumoorthy, unpublished results. From molecular modeling studies based upon high resolution chemical probe data, we have proposed a structural model for the complex of four human p53DBD peptides with thep21/waf1/cip1 response element. This model has provided a unique insight into the possible roles of DNA flexibility in the sequence specificity of p53 binding and has provided a rationale for the requirement of DNA bending both in the full tetrapeptide complex and in the individual decameric half-sites (22Nagaich A.K. Zhurkin V.B. Sakamoto H. Gorin A.A. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. J. Biol. Chem. 1997; 272: 14830-14841Google Scholar). However, none of these prior studies have addressed the relationship between DNA bending and binding affinity in p53 nucleoprotein complexes. In the present work, to further explore the relationships between DNA bending and p53 function, we have used circular permutation gel retardation and quantitative gel band shift assays to study the gel mobility retardation pattern of five biologically important binding sites: the p53 consensus binding sequence, a symmetric 20-bp binding sequence, the p21/waf1/cip1 response element, the ribosomal gene cluster (RGC) sequence, and the SV40 replication origin sequence (23Liu-Johnson H.-N. Gartenberg M.R. Crothers D.M. Cell. 1986; 47: 995-1005Google Scholar, 24Namba H. Hara T. Tukazaki T. Migita K. Ishikawa N. Ito K. Nagataki S. Yamashita S. Cancer Res. 1995; 55: 2075-2080Google Scholar, 25Kern S.E. Pietenpol J.A. Thiagalingam S. Seymour A. Kinzler K.W. Vogelstein B. Science. 1992; 256: 827-830Google Scholar, 26Funk W.D. Pak D.T. Karas R.H. Wright W.E. Shay J.W. Mol. Cell. Biol. 1992; 12: 2866-2871Google Scholar, 27Bargonetti J. Reynisdottir I. Friedman P.N. Prives C. Genes & Dev. 1992; 6: 1886-1898Google Scholar). The accuracy of bend angles estimated using circular permutation assays has been questioned recently in the case of several bZip proteins including Fos and Jun (28Kerppola T.K. Curran T. Mol. Cell. Biol. 1993; 13: 5479-5489Google Scholar, 29Sitlani A. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3248-3252Google Scholar) (reviewed by Hagerman (30Hagerman P.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9993-9996Google Scholar)) whose elongated shape and leucine zipper region differentiates them from most globular proteins. On the other hand, circular permutation assays provide bending angles that are in excellent agreement with results obtained from phasing and cyclization experiments and from x-ray crystallography for many globular proteins such as CAP, Cro, and the TATA-binding protein, and this method has been widely used to estimate DNA bending in such systems (31VanderVliet P.C. Verrijzer C.P. BioEssays. 1993; 15: 25-32Google Scholar, 32Starr D.B. Hoopes B.C. Hawley D.K. J. Mol. Biol. 1995; 250: 434-446Google Scholar). Since the p53DBD peptide has a similar globular conformation (18Cho Y.J. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Google Scholar), we believe that the circular permutation assay provides a satisfactory approximation to the true induced bending angles for this peptide bound to the various DNA response elements investigated in this work. Our data clearly show that the p53DBD binds with all of these binding sites cooperatively as a tetrapeptide and induces bending of the DNA. However, the bending angle varies considerably with different response elements over the range of 52–25°. Response elements having a d(CATG) sequence at the junctions of their consensus pentamers,i.e. the p53 consensus and symmetric sites and thep21/waf1/cip1 response element, are bent by ∼50°, whereas bending is much less (∼37-∼25°) in the case of the RGC and SV40 response elements, both of which have a d(CTTG) sequence at the pentamer junctions. Our results confirm an earlier report that p53DBD binds response elements cooperatively as a tetrapeptide and bends the DNA (21Balagurumoorthy P. Sakamoto H. Lewis M.S. Zambrano N. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8591-8595Google Scholar). In this earlier work, cyclization studies were used to estimate a bending angle of ∼50° in the p21/waf1/cip1 response element; the excellent agreement with the present result for this binding site provides further justification for the use of circular permutation to quantitate p53DBD DNA bending. We also find a positive correlation between the observed bending angles and binding affinities,i.e. the response elements that show higher bending angles also show higher p53DBD binding affinities, while response elements showing lower bending angles show much less binding affinity. A direct correlation between the binding affinity of p53 with various response elements and transcription activation has been observed by Kernet al. (25Kern S.E. Pietenpol J.A. Thiagalingam S. Seymour A. Kinzler K.W. Vogelstein B. Science. 1992; 256: 827-830Google Scholar). Furthermore, p53 consensus and RGC response elements have been characterized as distinctly different p53 binding elements; the former binds with certain p53 mutants and enhances transcriptional activity of the reporter genes, while the later does not bind with mutants (33Prives C. Manfredi J.J. Genes & Dev. 1993; 7: 529-534Google Scholar, 34McClure W.R. Annu. Rev. Biochem. 1985; 54: 171-204Google Scholar, 35Zhang W. Shay J.W. Diesseroth A. Cancer Res. 1993; 53: 4272-4775Google Scholar). Thus, our findings suggest that DNA bending may be a ubiquitous feature of p53DBD-DNA complexes irrespective of the DNA sequence and imply that the energetics of DNA bending may contribute significantly to the binding affinity of p53. In this event, the change in free energy associated with DNA bending in the p53-DNA complex may fine tune the transcriptional activation of p53-regulated genes. A human p53 cDNA clone encoding amino acid residues 96–308 was amplified by polymerase chain reaction using p53-specific primers 5′-ATATCATATGGTCCCTTCCCAGAAAACCTA-3′ and 5′-ATATGGATCCTCACAGTGCTCGCTTAGTGCTC-3′. The amplified product was cloned in the pET12a expression vector (Novagen), and the core DNA binding domain was overproduced in Escherichia coli BL21 (DE3). The cells were incubated at 37 °C until anA600 of 0.6–1.0 was attained, and 0.25 mm isopropyl β-d-thiogalactoside was added to induce the expression of the recombinant protein. Cells were harvested after 2 h by centrifugation, lysed in a French press, and sonicated for 2 min in 40 mm MES, pH 6.0, 100 mm NaCl, 5 mm dithiothreitol. The soluble fraction was loaded onto a Resource S column (Pharmacia Biotech Inc.) in 40 mm MES, pH 6.0, 5 mm dithiothreitol and was eluted in a 0–400 mm NaCl gradient. The pooled fractions were precipitated by ammonium sulfate addition to 80% saturation and purified further on a Sephadex 75 HR gel filtration column (Pharmacia) in 50 mm bis-Tris propane-HCl, pH 6.8, 100 mm NaCl, 1 mm dithiothreitol. The purified p53DBD was checked on an SDS-polyacrylamide gel for purity. All of the oligonucleotides used in this study contained a 20-bp consensus p53 binding site in a 30-mer oligonucleotide having XbaI and SalI cohesive termini (Fig. 1 A). The oligonucleotides were synthesized using β-cyanoethyl phosphoramidite chemistry and purified on polyacrylamide gel using standard procedures (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 13.78-13.95Google Scholar). These oligonucleotides were directionally subcloned at the XbaI and SalI restriction sites of DNA bending vector pBend3 (37Kim J. Zweib C. Wu C. Adhya S. Gene ( Amst. ). 1989; 85: 15-23Google Scholar). The recombinant plasmids were prepared by an alkaline lysis procedure and sequenced to confirm the size and orientation of the insert. The purified plasmids were digested with MluI,NheI, SpeI, EcoRV, StuI,NcoI, and BamHI restriction enzymes to generate DNA fragments having a circularly permuted p53 consensus binding site (Fig. 1 B). The digested fragments were purified on a low melting agarose gel using a Geneclean kit (Bio101). The cohesive end fragments were labeled with [α-32P]dCTP and the Klenow fragment, whereas the blunt-ended fragments were dephosphorylated with calf intestinal alkaline phosphatase and then labeled with [γ-32P]ATP and polynucleotide kinase. The labeled DNA fragments were again purified on a native polyacrylamide gel to eliminate traces of free label and other undesired fragments. The labeled DNA fragments were mixed with poly(dI-dC) (200 ng) and incubated with the purified p53DBD in the DNA binding buffer (50 mm bis-Tris propane, HCl, pH 6.8, 1 mmdithiothreitol, and 50 mm NaCl) (10 μl) at 4 °C for 40 min. The amount of p53DBD was adjusted such that no more than 50% of the DNA was retarded due to complex formation. The reactions were mixed with 15% Ficoll (2 μl) and loaded onto 5% native polyacrylamide gel. The gels were run in 0.25 × TBE at 150 V for about 1.5 h, dried, and autoradiographed. p53DBD-induced DNA bending was measured by electrophoretic mobility shift assay using DNA fragments containing circularly permuted binding sites. The relative mobility (RF) of the p53DBD-DNA complexes was defined as follows, RF=CmcomplexCmfreeEquation 1 where Cm represents the migration of the fragments in the gel in centimeters. The lengths of the different probes varied from 145 to 149 bp due to the sequence of the pBend3 polylinker and the difference in labeling procedures (end filling versuskinasing). To adjust for the slight differences in the probe length, the relative mobilities of the p53DBD-DNA complexes were normalized against highest mobility probe. The average relative mobility from three different experiments was plotted as a function of the fractional displacement, defined as the distance in base pairs from the center of the p53DBD response element to the 5′-end of the noncoding strand of the DNA fragment divided by the total length of the fragment. These data, when plotted as relative mobility versus fractional displacement, could be approximated by a least squares fit to a parabolic function of the form y =ax 2 + bx + c. The bending center (y = minimum) was determined by setting dy/dx to 0 and solving for x. To determine bending angles, we have used the mathematical treatment derived by Ferrari et al., where the bend angle θ is determined from the values for a, b, andc, taken from the least squares fit to the quadratic equation, using the following equation: a or (−b) = 2c(1 + cosθ). With this method, the values of θ using a or −b should be identical, and their comparison offers a means for estimating the error in the measured bending angle. The angle of deviation from linearity, α, is related to θ by the equation α = 180° − θ (38Ferrari S. Harley V.R. Pontiggia A. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1992; 11: 4497-4506Google Scholar). The magnitude of the protein-induced DNA bending was also calculated by a semiempirical equation described by Thompson and Landy (39Thompson J.F. Landy A. Nucleic Acids Res. 1988; 16: 9687-9705Google Scholar). μMμE=coskα2Equation 2 where μM and μE are the mobility of the protein-DNA complex with the binding site at the middle and end of the DNA fragment, respectively, k is a coefficient that depends upon the electrophoresis conditions, and α is the angle of the protein-induced DNA bend. A k value of ∼1 was obtained for our gel electrophoretic conditions using a calibration oligonucleotide having an intrinsic bend of 54°. The magnitude of the p53DBD-induced bend angles were determined using this same value of k. DNA binding affinities of the p53DBD with different response elements were assayed using electrophoretic mobility shift assays. The 30-mer oligonucleotide duplexes were labeled with [γ-32P]ATP and polynucleotide kinase, purified on a native polyacrylamide gel, and quantitated by UV spectrophotometry, using ε values of 15,500m−1 cm−1, 8,500m−1 cm−1, 12,500m−1 cm−1, and 7,500m−1 cm−1 for the A, T, G, and C residues, respectively. The protein concentration was measured spectrophotometrically using a Bradford protein assay kit (Bio-Rad). The specific DNA binding activity of these preparations was found to be ∼90%. Each labeled oligonucleotide (∼15 pmol) was mixed with poly(dI-dC) (200 ng) and incubated with increasing amounts of the known concentrations of active p53DBD in DNA binding buffer (10 μl) for 40 min at 4 °C. The samples were mixed with 15% Ficoll (2 μl) to facilitate gel loading and electrophoresed on a pre-electrophoresed 7% polyacrylamide gel (29:1, acrylamide:bis) in 0.25 × TBE for 2 h at 8 V/cm at 4 °C. The loading was done rapidly to minimize the equilibration of the complex with the running buffer. The gels were then transferred to Whatman filter paper, dried under vacuum, and exposed to the x-ray film. The same gel was used to quantitate the free and the bound DNA using a PhosphorImager (Bio-Rad). The exposure was adjusted to achieve a linear response of radioactivity with measured band intensities. Radioactivity scattered between the bound and the free bands was counted with the free probe. The quantitation of the different bands was carried out using Molecular Analyst, a Bio-Rad gel analysis software using standard procedures (40Carey J. Methods Enzymol. 1991; 208: 103-117Google Scholar). The fraction of the free and bound molecules at each protein concentration was calculated by dividing the optical density of the band by the sum of the optical densities in all the bands in the same lane. Since the concentration of the DNA in these reaction mixtures was always ≤15 pmol, it was assumed that [p53DBD]total ≈ [p53DBD]free, so the protein concentration required for half maximal binding is very close toKd(app). The data were plotted in the form of a Bjerrum plot of the fraction of free DNA versusthe log of protein concentration, and the dissociation constants (Kd) were determined as the protein concentration at which half of the free DNA was bound. In determiningKd(app), we chose the disappearance of the free DNA versus protein concentration rather than the appearance of the complex bands to account for any dissociation of the complex during the running of the gel. The free energy of binding was calculated using the relation ΔG 0=−RTln K(app) at 4 °C, and the free energy of bending was calculated from the following equation (23Liu-Johnson H.-N. Gartenberg M.R. Crothers D.M. Cell. 1986; 47: 995-1005Google Scholar). ΔGbend=0.014£(bp)(Δθ)2(kcal/mol)Equation 3 In Equation 3, Δθ is the bending angle in degrees induced by binding of protein to DNA, and £(bp) is the number of base pairs involved in the complex. The DNA is assumed to have a persistence length of 150 bp (41Hagerman P.J. Biopolymers. 1981; 20: 1503-1535Google Scholar, 42Cairney K.L. Harrington R.E. Biopolymers. 1982; 21: 923-934Google Scholar). We have described the cooperative binding of the p53DBD with the response elements using the method of Senear and Brenowitz (43Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Google Scholar) for the two-site cooperative binding of protein with the DNA as described below. In Scheme FS1, k1 andk2 are the intrinsic binding constants to sites 1 and 2, respectively, and k12 is the cooperativity coefficient. The fraction of the molecules [Θ]i that are free, single, and doubly liganded can be written, respectively, as follows, Θ0=11+(k1+k2)[X]+(k1k2k12)[X2]Equation 4 Θ1=(k1+k2)[X]21+(k1+k2)[X]+(k1k2k12)[X2]Equation 5 Θ2=(k1k2k12)[X]21+(k1+k2)[X]+(k1k2k12)[X2]Equation 6 where X is the concentration of the free protein. Plots of the fraction of free and bound DNA versus the log of protein concentrations were fitted to Equations 4 and 6, respectively, and the cooperativity parameter was calculated as described by Senear and Brenowitz (43Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Google Scholar). Using T4 DNA ligase-mediated cyclization assays, our laboratory has previously shown that the p53DBD binds the response elements cooperatively as a tetrapeptide and bends the DNA (21Balagurumoorthy P. Sakamoto H. Lewis M.S. Zambrano N. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8591-8595Google Scholar). High resolution chemical footprinting has shown that four p53DBD peptides bind the full 20-bp p21/waf1/cip1 response element DNA in a staggered array, and molecular modeling based upon protein-DNA contacts identified by base-specific chemical probes has suggested that the bound p53DBD must bend the response element DNA to relieve two types of steric clashes among the bound peptides (22Nagaich A.K. Zhurkin V.B. Sakamoto H. Gorin A.A. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. J. Biol. Chem. 1997; 272: 14830-14841Google Scholar). In the present work, we have used the circular permutation polyacrylamide gel retardation assay (44Wu H.-M. Crothers D.M. Nature. 1984; 308: 509-513Google Scholar) to confirm and amplify these findings and to examine the DNA bending relationships among different p53 response elements. The gel retardation assay is based on the assumption that static bending in DNA leads to a reduction in gel mobility as predicted by the Lumpkin-Zimm reptation model, in which the mobility of DNA through a gel is a quadratic function of its mean square end-to-end distance (45Lumpkin O.J. Zimm B.H. Biopolymers. 1982; 21: 2315-2316Google Scholar, 46Levene S.D. Zimm B.H. Science. 1989; 245: 396-399Google Scholar). Uniform length DNA fragments show increasingly anomalous electrophoretic mobility on polyacrylamide gels, as the bending locus is moved from the ends to the center of the DNA fragment. Although the relationship between electrophoretic mobility and conformation for protein-DNA complexes is complex, a comparison of the mobilities of circularly permuted p53DBD-DNA complexes allows a relatively precise determination of the DNA bending locus and an estimate of the bending angle (38Ferrari S. Harley V.R. Pontiggia A. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1992; 11: 4497-4506Google Scholar, 47Nardulli A.M. Shapiro D.J. Mol. Cell. Biol. 1992; 12: 2037-2042Google Scholar). Gel mobility retardation data for all circularly permuted fragments containing p53DBD bound to p53 consensus, p21/waf1/cip1, symmetric, RGC and SV40 response elements (Fig. 1) are shown in Fig. 2, A–E. Corresponding plots of relative gel mobility as functions of flexure displacements of different DNA sequences are shown in Fig. 2, F–J. The unbound fractions of the circularly permuted DNA fragments in all of the gels (bands marked as F in Fig. 2, A–E) show similar mobilities on the polyacrylamide gels, whereas the mobilities for bound fractions (bands marked as C) are clearly anomalous. The mobilities are maximum when the bound response elements are near the ends of the fragments (lanes marked as M andB) and minimum when they are near the centers (lanes marked as E). Intermediate mobilities are observed at intermediate positions (lanes marked as Nh, Sp, S, and Nc). In particular, the bound fractions for the p53 consensus, p21/waf1/cip1, and symmetric response elements (Fig. 2, F–H) show much higher migration anomalies compared with RGC and SV40 (Fig. 2, I and J), as is evident also from a comparison of calculated α values for these sequences. Nevertheless, all of the response elements investigated show gel migration anomalies characteristic of protein-induced DNA bending, and the smallest bending angle observed, α = 25° for the SV40 site (Fig. 4, E and J), still represents considerable DNA bending in this complex.Figure 4Titration curves resolved from the gel mobility shift experiments of p53DBD with p53 consensus sequence (A), p21/waf1/cip1 (B), symmetric sequence (C), RGC sequence (D), and SV40 binding sequence (E). The fraction of free DNA averaged from three independent experiments versus the log of p53DBD concentrations has been plotted. The solid linesindicate the theoretical curve generated by fitting the data to Equation 4 (see "Materials and Methods").View Large Image Figure ViewerDownload (PPT) The data of Fig. 2 support the interpretation that the observed electrophoretic mobilities depend upon the position of the bound p53DBD within each DNA fragment and hence that the mobility anomalies must be due to DNA bending. However, to ensure that the gel conditions used were not a factor in the apparent differences in mobility, identical experiments were also carried out using 4–1