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• W2040256004 abstract "The TATA-binding protein (TBP) recognizes the TATA box element of transcriptional promoters and recruits other initiation factors. This essential protein binds selectively to cisplatin-damaged DNA. Electrophoretic mobility shift assays were performed to study the kinetics of TBP binding both to the TATA box and to cisplatin-damaged DNA in different sequence contexts. TBP binds with high affinity (Kd = 0.3 nm) to DNA containing site-specific cisplatin 1,2-intrastrand d(GpG) cross-links. The kon and koff values for the formation of these TBP complexes are 1–3 × 105m−1 s−1 and ∼1–5 × 10−4 s−1, respectively, similar to the corresponding values for the formation of a TBP-TATA box complex. In electrophoretic mobility shift assay competition assays, cisplatin-damaged DNA extensively sequesters TBP from its natural binding site, the TATA box. Nine DNA probes were prepared to determine the flanking sequence dependence of TBP binding to cisplatin-modified DNA. TBP clearly displays sequence context selectivity for platinated DNA, very similar to but not as dramatic as that of the high mobility group protein HMGB1. When TBP was added to an in vitronucleotide excision repair assay, it specifically shielded cisplatin-modified 1,2-(GpG) intrastrand cross-links from repair. These results indicate that TBP is likely to be a key protein in mediating the cytotoxicity of cisplatin. The TATA-binding protein (TBP) recognizes the TATA box element of transcriptional promoters and recruits other initiation factors. This essential protein binds selectively to cisplatin-damaged DNA. Electrophoretic mobility shift assays were performed to study the kinetics of TBP binding both to the TATA box and to cisplatin-damaged DNA in different sequence contexts. TBP binds with high affinity (Kd = 0.3 nm) to DNA containing site-specific cisplatin 1,2-intrastrand d(GpG) cross-links. The kon and koff values for the formation of these TBP complexes are 1–3 × 105m−1 s−1 and ∼1–5 × 10−4 s−1, respectively, similar to the corresponding values for the formation of a TBP-TATA box complex. In electrophoretic mobility shift assay competition assays, cisplatin-damaged DNA extensively sequesters TBP from its natural binding site, the TATA box. Nine DNA probes were prepared to determine the flanking sequence dependence of TBP binding to cisplatin-modified DNA. TBP clearly displays sequence context selectivity for platinated DNA, very similar to but not as dramatic as that of the high mobility group protein HMGB1. When TBP was added to an in vitronucleotide excision repair assay, it specifically shielded cisplatin-modified 1,2-(GpG) intrastrand cross-links from repair. These results indicate that TBP is likely to be a key protein in mediating the cytotoxicity of cisplatin. TATA-binding protein electrophoretic mobility shift assay high mobility group high mobility group box protein 1 cis-diamminedichloroplatinum(II) replication protein A base pair(s) high performance liquid chromatography dithiothreitol nucleotide excision repair cell-free extract major late promoter After the discovery of the anticancer activity of cisplatin, many studies have focused on elucidating its mechanism of action (1Jamieson E.R. Lippard S.J. Chem. Rev. 1999; 99: 2467-2498Crossref PubMed Scopus (2638) Google Scholar, 2Cohen S.M. Lippard S.J. Prog. Nucleic Acid Res. Mol. Biol. 2001; 67: 93-130Crossref PubMed Scopus (555) Google Scholar). The formation of covalent cisplatin-DNA adducts, especially the 1,2-intrastrand d(GpG) cross-link, correlates with the cytotoxicity of the drug (1Jamieson E.R. Lippard S.J. Chem. Rev. 1999; 99: 2467-2498Crossref PubMed Scopus (2638) Google Scholar, 2Cohen S.M. Lippard S.J. Prog. Nucleic Acid Res. Mol. Biol. 2001; 67: 93-130Crossref PubMed Scopus (555) Google Scholar, 3Zamble D.B. Lippard S.J. Lippert B. Cisplatin Chemistry and Biochemistry of a Leading Anticancer Drug. Verlag Helvetica Chimica Acta, Zurich1999: 73-110Google Scholar). Attention now focuses on understanding how cells react to the presence of the cisplatin-DNA lesions and, using this information, designing more effective anticancer treatments. Upon cisplatin binding, the DNA duplex is bent and unwound, and the minor groove becomes wide and shallow. These structural changes inhibit essential DNA metabolic processes such as replication and transcription (4Comess K.M. Burstyn J.N. Essigmann J.M. Lippard S.J. Biochemistry. 1992; 31: 3975-3990Crossref PubMed Scopus (147) Google Scholar, 5Suo Z. Lippard S.J. Johnson K.A. Biochemistry. 1999; 38: 715-726Crossref PubMed Scopus (77) Google Scholar, 6Mello J.A. Lippard S.J. Essigmann J.M. Biochemistry. 1995; 34: 14783-14791Crossref PubMed Scopus (97) Google Scholar, 7Jordan P. Carmo-Fonseca M. Nucleic Acids Res. 1998; 26: 2831-2836Crossref PubMed Scopus (110) Google Scholar). The distorted DNA duplex can also interact with a number of cellular proteins (1Jamieson E.R. Lippard S.J. Chem. Rev. 1999; 99: 2467-2498Crossref PubMed Scopus (2638) Google Scholar, 2Cohen S.M. Lippard S.J. Prog. Nucleic Acid Res. Mol. Biol. 2001; 67: 93-130Crossref PubMed Scopus (555) Google Scholar, 8Kartalou M. Essigmann J.M. Mutat. Res. 2001; 478: 1-21Crossref PubMed Scopus (336) Google Scholar, 9Zlatanova J. Yaneva J. Leuba S.H. FASEB J. 1998; 12: 791-799Crossref PubMed Scopus (117) Google Scholar), an activity postulated to mediate the processing of cisplatin-DNA lesions (1Jamieson E.R. Lippard S.J. Chem. Rev. 1999; 99: 2467-2498Crossref PubMed Scopus (2638) Google Scholar, 2Cohen S.M. Lippard S.J. Prog. Nucleic Acid Res. Mol. Biol. 2001; 67: 93-130Crossref PubMed Scopus (555) Google Scholar, 3Zamble D.B. Lippard S.J. Lippert B. Cisplatin Chemistry and Biochemistry of a Leading Anticancer Drug. Verlag Helvetica Chimica Acta, Zurich1999: 73-110Google Scholar, 10Hoffmann J.S. Locker D. Villani G. Leng M. J. Mol. Biol. 1997; 270: 539-543Crossref PubMed Scopus (32) Google Scholar, 11Brown S.J. Kellett P.J. Lippard S.J. Science. 1993; 261: 603-605Crossref PubMed Scopus (181) Google Scholar, 12Huang J.C. Zamble D.B. Reardon J.T. Lippard S.J. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10394-10398Crossref PubMed Scopus (342) Google Scholar, 13He Q. Liang C.H. Lippard S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5768-5772Crossref PubMed Scopus (204) Google Scholar, 14Kasparkova J. Pospisilova S. Brabec V. J. Biol. Chem. 2001; 276: 16064-16069Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The identification and characterization of proteins that bind selectively to cisplatin-damaged DNA has therefore become one of the main thrusts of research in this field. The TATA-binding protein (TBP)1 is a key component of transcription factor IID, which is required for transcription initiation of all three eukaryotic RNA polymerases (15Cormack B.P. Struhl K. Cell. 1992; 69: 685-696Abstract Full Text PDF PubMed Scopus (277) Google Scholar). As the first step in the process, TBP recognizes a TATA box element located ∼30 base pairs (bp) upstream from the transcription start site and eventually recruits other transcription factors. Structural analyses of several TBP-TATA box complexes reveal that TBP binds at a widened minor groove and bends the duplex DNA toward the major groove by intercalation of two pairs of phenylalanine residues (16Patikoglou G.A. Kim J.L. Sun L. Yang S.H. Kodadek T. Burley S.K. Genes Dev. 1999; 13: 3217-3230Crossref PubMed Scopus (246) Google Scholar, 17Nikolov D.B. Chen H. Halay E.D. Hoffman A. Roeder R.G. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4862-4867Crossref PubMed Scopus (257) Google Scholar, 18Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Crossref PubMed Scopus (1016) Google Scholar). Because sequence-specific DNA-binding proteins usually reside in the major groove, the minor groove binding by TBP was initially puzzling. Subsequent studies demonstrated that the flexibility of the TATA box element primarily determines its binding affinity to TBP (19Juo Z.S. Chiu T.K. Leiberman P.M. Baikalov I. Berk A.J. Dickerson R.E. J. Mol. Biol. 1996; 261: 239-254Crossref PubMed Scopus (287) Google Scholar, 20Grove A. Galeone A. Yu E. Mayol L. Geiduschek E.P. J. Mol. Biol. 1998; 282: 731-739Crossref PubMed Scopus (49) Google Scholar). In addition to the specificity of its DNA binding, TBP has distinctive DNA binding kinetics. The formation of the TBP-DNA complex is characterized by relatively slow on and off rates (21Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar). TBP binds selectively to cisplatin-damaged DNA (22Vichi P. Coin F. Renaud J.P. Vermeulen W. Hoeijmakers J.H.J. Moras D. Egly J.M. EMBO J. 1997; 16: 7444-7456Crossref PubMed Scopus (155) Google Scholar). The sequestration of TBP by cisplatin DNA adducts inhibits transcription in vitro, which could be restored in a reconstituted system by addition of extra TBP (22Vichi P. Coin F. Renaud J.P. Vermeulen W. Hoeijmakers J.H.J. Moras D. Egly J.M. EMBO J. 1997; 16: 7444-7456Crossref PubMed Scopus (155) Google Scholar). Transcription inhibition by exogenously added cisplatin-damaged DNA has also been reported (23Cullinane C. Mazur S.J. Essigmann J.M. Phillips D.R. Bohr V.A. Biochemistry. 1999; 38: 6204-6212Crossref PubMed Scopus (76) Google Scholar). More recently, enhanced binding of TBP to the TATA element containing flanking cisplatin 1,2-intrastrand cross-links was demonstrated (24Cohen S.M. Jamieson E.J. Lippard S.J. Biochemistry. 2000; 39: 8259-8265Crossref PubMed Scopus (46) Google Scholar). This series of experiments suggests a possible role for TBP-cisplatin-DNA ternary complexes in mediating the anticancer activity of the drug. Because a variety of proteins interact with cisplatin-DNA adducts (9Zlatanova J. Yaneva J. Leuba S.H. FASEB J. 1998; 12: 791-799Crossref PubMed Scopus (117) Google Scholar), many studies have focused on determining their binding affinities (25Pil P.M. Lippard S.J. Science. 1992; 256: 234-237Crossref PubMed Scopus (512) Google Scholar, 26Dunham S.U. Lippard S.J. Biochemistry. 1997; 36: 11428-11436Crossref PubMed Scopus (78) Google Scholar, 27McA'Nulty M.M. Whitehead J.P. Lippard S.J. Biochemistry. 1996; 35: 6089-6099Crossref PubMed Scopus (53) Google Scholar, 28Hey T. Lipps G. Krauss G. Biochemistry. 2001; 40: 2901-2910Crossref PubMed Scopus (51) Google Scholar, 29Ohndorf U.M. Whitehead J.P. Raju N.L. Lippard S.J. Biochemistry. 1997; 36: 14807-14815Crossref PubMed Scopus (51) Google Scholar, 30Chow C.S. Whitehead J.P. Lippard S.J. Biochemistry. 1994; 33: 15124-15130Crossref PubMed Scopus (95) Google Scholar). To evaluate the relative importance of the different protein-platinated DNA interactions in the cell, however, detailed kinetic parameters are also required. Despite the importance of such information, few attempts have been made to examine the kinetics of protein binding to cisplatin-damaged DNA (31Jamieson E.R. Jacobson M.P. Barnes C.M. Chow C.S. Lippard S.J. J. Biol. Chem. 1999; 274: 12346-12354Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 32Jamieson E.R. Lippard S.J. Biochemistry. 2000; 39: 8426-8438Crossref PubMed Scopus (28) Google Scholar, 33Patrick S.M. Turchi J.J. J. Biol. Chem. 2001; 276: 22630-22637Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The interactions between the two domains (A and B) of HMGB1 with cisplatin-modified DNA were investigated by using stopped-flow fluorescence and fluorescence resonance energy transfer methods (31Jamieson E.R. Jacobson M.P. Barnes C.M. Chow C.S. Lippard S.J. J. Biol. Chem. 1999; 274: 12346-12354Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 32Jamieson E.R. Lippard S.J. Biochemistry. 2000; 39: 8426-8438Crossref PubMed Scopus (28) Google Scholar). In addition, a stopped-flow kinetic analysis was performed for replication protein A (RPA) binding to cisplatin-damaged DNA (33Patrick S.M. Turchi J.J. J. Biol. Chem. 2001; 276: 22630-22637Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In the present study we employed electrophoretic mobility shift assays (EMSAs) to examine the kinetics of TBP binding to cisplatin-damaged DNA and, for comparison purposes, the TATA box. TBP binding to DNA containing a site-specific 1,2-intrastrand d(GpG) cross-link was evaluated by using several platinum-modified DNA probes with various flanking sequences. We also performed an in vitro repair assay to examine the effect of TBP in modulating nucleotide excision repair (NER) of platinated DNA. The results provide strong evidence for a biological role of TBP in mediating the cytotoxicity of cisplatin. The C-terminal DNA binding domain of recombinant yeast TBP was provided by Dr. S. M. Cohen in our laboratory and prepared as described previously (24Cohen S.M. Jamieson E.J. Lippard S.J. Biochemistry. 2000; 39: 8259-8265Crossref PubMed Scopus (46) Google Scholar). The concentration of active TBP was determined as reported in the literature (34Hahn S. Buratowski S. Sharp P.A. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5718-5722Crossref PubMed Scopus (219) Google Scholar). Table I lists the 25-bp oligonucleotides used in this study together with their abbreviations. The synthesis, platination, and purification of site-specifically platinated single-stranded oligonucleotides were carried out as described previously (26Dunham S.U. Lippard S.J. Biochemistry. 1997; 36: 11428-11436Crossref PubMed Scopus (78) Google Scholar). Platinated top strands, (5′-CCTCTCCTCTCN1G*G*N2 TCTTCTCTCC-3′, N1 and N2 = A, T, or C), where the asterisks indicate the formation of Pt-N(7) bonds, were annealed with their complementary bottom strands in 10 mm Tris (pH 7.0), 50 mm NaCl, and 10 mm MgCl2, heated to 90 °C, and slowly cooled to 4 °C over several hours. The resulting cisplatin-modified duplex probes were purified by ion-ex- change HPLC using a Dionex DNApac PA-100 column. These oligonucleotides were desalted by dialysis and concentrated to 5–10 μm. The TATAMLP probe (Table I) was prepared by the same method except for the platination step. The TATA element of adenovirus major late promoter, one of the strongest promoters, was used for this study. The purity of the platinated single-stranded DNA was confirmed by analytical HPLC. Atomic absorption spectroscopy combined with UV-visible spectroscopy and electrospray mass spectrometry verified the existence of singly platinated probes (see the supplemental material on-line).Table IDuplex DNA probes and abbreviationsAbbreviationProbes1-aBoldface font denotes the TATA box, cisplatin d(GpG) lesion (N1GGN2), and flanking sequences.TATAMLP5′-AAGGGGGGCTATAAAAGGGGGTGGG-3′3′-TTCCCCCCGATATTTTCCCCCACCC-5′AGGA5′-CCTCTCCTCTCAGGATCTTCTCTCC-3′3′-GGAGAGGAGAGTCCTAGAAGAGAGG-5′AGGT5′-CCTCTCCTCTCAGGTTCTTCTCTCC-3′3′-GGAGAGGAGAGTCCAAGAAGAGAGG-5′AGGC5′-CCTCTCCTCTCAGGCTCTTCTCTCC-3′3′-GGAGAGGAGAGTCCGAGAAGAGAGG-5′TGGA5′-CCTCTCCTCTCTGGATCTTCTCTCC-3′3′-GGAGAGGAGAGACCTAGAAGAGAGG-5′TGGT5′-CCTCTCCTCTCTGGTTCTTCTCTCC-3′3′-GGAGAGGAGAGACCAAGAAGAGAGG-5′TGGC5′-CCTCTCCTCTCTGGCTCTTCTCTCC-3′3′-GGAGAGGAGAGACCGAGAAGAGAGG-5′CGGA5′-CCTCTCCTCTCCGGATCTTCTCTCC-3′3′-GGAGAGGAGAGGCCTAGAAGAGAGG-5′CGGT5′-CCTCTCCTCTCCGGTTCTTCTCTCC-3′3′-GGAGAGGAGAGGCCAAGAAGAGAGG-5′CGGC5′-CCTCTCCTCTCCGGCTCTTCTCTCC-3′3′-GGAGAGGAGAGGCCGAGAAGAGAGG-5′1-a Boldface font denotes the TATA box, cisplatin d(GpG) lesion (N1GGN2), and flanking sequences. Open table in a new tab All platinated and TATAMLP duplex probes (∼10 pmol) were radioactively labeled at their 5′-ends by using 50 μCi of [γ-32P]ATP (PerkinElmer Life Sciences) and 20 units of polynucleotide kinase (New England Biolabs). Labeled probes were separated from small nucleotides by passage through G-25 Sephadex Quickspin columns (Roche Molecular Biochemicals). For each binding reaction, DNA probes (0.5–2 nm, ∼10,000 cpm) and the indicated concentrations of TBP were mixed and incubated in a buffer solution containing 60 mm KCl, 20 mm Tris (pH 7.9), 5.0 mm MgCl2, 10 mmdithiothreitol, 0.2 mg/ml bovine serum albumin, and 10% glycerol (24Cohen S.M. Jamieson E.J. Lippard S.J. Biochemistry. 2000; 39: 8259-8265Crossref PubMed Scopus (46) Google Scholar). After incubation at 30 °C for 30 min, binding mixtures were directly loaded onto 6% native polyacrylamide gels and electrophoresed for 1.5 h at 150 V in 25 mm Tris (pH 7.9), 190 mm glycine, 1.0 mm EDTA, and 4.0 mmMgCl2 running buffer. The gels were dried at 80 °C and then exposed to phosphorimaging plates for 15–20 h. Quantitative analysis was performed with a Bio-Rad GS-525 Molecular Imager employing Multi-analyst software (Bio-Rad). For the measurement of the protein-DNA association constants by kinetic methods, the increase in the amount of complex was monitored at various times after the addition of 5–10 nm TBP to ∼1 nm probe. As described previously (35Parvin J.D. McCormick R.J. Sharp P.A. Fisher D.E. Nature. 1995; 373: 724-727Crossref PubMed Scopus (172) Google Scholar), the raw data were fit to Equation 1, 1/[TBP]0ln([probe]0/{[probe]0−[complex]})=kontEq. 1 which is derived from the bimolecular binding equation under conditions of excess protein (36Riggs A.D. Bourgeois S. Cohn M. J. Mol. Biol. 1970; 53: 401-417Crossref PubMed Scopus (641) Google Scholar). The left side of the equation was plotted against time in s, and the kon value for each probe was obtained from the slope of the plot. In the equation, [TBP]0 and [probe]0 indicate the initial concentrations of TBP and DNA probe, respectively, and [complex] is the concentration of complex at each time point. To determine the dissociation rate constants (koff), ∼10 nm TBP and ∼1 nm probe were mixed at 30 °C for 30 min to reach the equilibrium state. Dissociation of the protein-DNA complexes was initiated by the addition of 200 ng of poly[dI·dC] at different time points to assure that all dissociation reactions were complete at the same time. The decrease in the amount of complex was followed over a 1- or 2-h time period. The data were fit to Equation 2 to obtain the first-order rate constant for the dissociation reaction,ln{[complex]/[complex]0}=−kofftEq. 2 where [complex] indicates the concentration of complex at time t, and [complex]0 is the complex concentration under the initial binding conditions. The natural logarithm of [complex] divided by [complex]0 was plotted versus time, and the negative slope of the fit provided the koff value. The dissociation constant,Kd, values were calculated from these results (Kd=kon/koff). Competitor DNA of varying concentrations was mixed with a fixed amount of TBP and radioactively labeled TATAMLP, and the disappearance of the TBP-TATAMLP complex was analyzed by EMSA. Competition assays were also used to obtain the Kdvalues for weak binding probes. In such a competition experiment, two binding reactions proceed simultaneously and follow the relationship in Equation 3 (37Lin S.Y. Riggs A.D. J. Mol. Biol. 1972; 72: 671-690Crossref PubMed Scopus (225) Google Scholar),θ=Pt(1−θ)Kt(1+Ct)/Kc+Tt(1−θ)Eq. 3 where Pt, Ct, and Tt represent the total concentrations of TBP, competitor, and TATAMLP, respectively. Kt and Kc are the respective dissociation constants of TATAMLP and competitor. At different Ct values, the binding fraction (θ) was determined. The Kd value can be calculated by using Equation 4, where C1/2 represents the concentration of competitor at θ = 1/2 (37Lin S.Y. Riggs A.D. J. Mol. Biol. 1972; 72: 671-690Crossref PubMed Scopus (225) Google Scholar).Kc=2KtC1/22Pt−Tt−2KtEq. 4 Whole cell-free extracts (CFE) from HeLa cells were prepared by a reported method (38Manley J.L. Fire A. Cano A. Sharp P.A. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3855-3859Crossref PubMed Scopus (735) Google Scholar) and stored at −80 °C. DNA duplexes 161 bp in length containing a site-specific cisplatin 1,2-d(GpG) or 1,3-d(GpTpG) cross-link with a radiolabeled phosphate located six or seven bases to the 5′ side of the cisplatin binding site were prepared as previously described (39Zamble D.B. Mu D. Reardon J.T. Sancar A. Lippard S.J. Biochemistry. 1996; 35: 10004-10013Crossref PubMed Scopus (311) Google Scholar, 40Huang J.C. Svoboda D.L. Reardon J.T. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3664-3668Crossref PubMed Scopus (379) Google Scholar). The probes contained three phosphorothioate residues at their 3′ ends to minimize nonspecific nuclease degradation. Whole cell extracts were incubated with damaged DNA in excision repair buffer (39Zamble D.B. Mu D. Reardon J.T. Sancar A. Lippard S.J. Biochemistry. 1996; 35: 10004-10013Crossref PubMed Scopus (311) Google Scholar), and excision fragments were resolved by 10% denaturing polyacrylamide gel electrophoresis. For the repair experiment in the presence of TBP, the protein was pre-incubated with the damaged DNA probe in excision repair buffer for 30 min on ice unless otherwise indicated. The extent of NER was measured by comparing the signal intensity corresponding to the 25–30-bp excised fragment with that of the entire lane. The small amount of DNA degradation due to nonspecific nuclease activity was subtracted from the repair signal by using the area corresponding to the 32–37-bp region as background (41Branum M.E. Reardon J.T. Sancar A. J. Biol. Chem. 2001; 276: 25421-25426Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). To optimize conditions for the EMSA experiments, probes of three different lengths (15, 25, and 35 bp) were investigated. The 15-bp probe showed weak binding affinity to TBP compared with longer probes. In addition, this short oligonucleotide was partially melted under the experimental conditions (data not shown). The 35-bp probe displayed a considerable level of nonspecific binding (data not shown). The 25-bp probe was stable under the experimental conditions and did not exhibit any nonspecific binding. Therefore, 25-bp probes were used for further studies. EMSA analyses were performed to investigate the kinetics of TBP binding to the TATA box (TATAMLP) and cisplatin-damaged DNA (AGGA, TableI). Fig.1 A shows EMSA data used to determine the association rate constants for two probes, and the data analysis is presented in Fig. 1 B. The bimolecular binding reactions reach the equilibrium state within 10 min for both probes. As shown in Fig. 1 A, however, the TBP-AGGA complex clearly forms more rapidly than the TBP-TATAMLP complex. Data collected within 2–3 min were fit to Equation 1, yielding konvalues of 1.3 × 105m−1s−1 for TBP binding to TATAMLP and 3.0 × 105m−1 s−1 for the AGGA probe (Fig. 1 B). The kon value for TATAMLP is in good agreement with published values, which fall in the range 1.0–3.0 × 105m−1s−1 (21Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar, 35Parvin J.D. McCormick R.J. Sharp P.A. Fisher D.E. Nature. 1995; 373: 724-727Crossref PubMed Scopus (172) Google Scholar, 42Petri V. Hsieh M. Jamison E. Brenowitz M. Biochemistry. 1998; 37: 15842-15849Crossref PubMed Scopus (45) Google Scholar, 43Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Mol. Biol. 1998; 277: 1015-1031Crossref PubMed Scopus (61) Google Scholar, 44Wolner B.S. Gralla J.D. J. Biol. Chem. 2001; 276: 6260-6266Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). After the addition of excess of poly[dI·dC] to the TBP-DNA complex, decreased amounts of the complex were detected (Fig.2). The dissociation reaction data were fit to Equation 2. The AGGA and TATAMLP probes dissociate from TBP with half-lives, t12, of 120 min and 190 min, respectively. Although TBP associates with AGGA more rapidly than with TATAMLP, the latter probe has a slightly lower off-rate (Table II).Table IIKinetic and thermodynamic parameters for TBP binding to each probeProbekonkoffKdm−1s−1s−1nmTATAMLP1.3 ± 0.2 × 1050.57 ± 0.05 × 10−40.44AGGA3.0 ± 0.5 × 1050.89 ± 0.09 × 10−40.30AGGT2.1 ± 0.2 × 1050.96 ± 0.04 × 10−40.46AGGC2.3 ± 0.1 × 1051.1 ± 0.1 × 10−40.48TGGA1.6 ± 0.2 × 1054.5 ± 0.0 × 10−42.8TGGT1.5 ± 0.1 × 1053.0 ± 0.1 × 10−42.0TGGC12 ± 1.02-aMeasured by competition assay (see “Experimental Procedures”).CGGA1.3 ± 0.1 × 1052.6 ± 0.2 × 10−42.0CGGT1.2 ± 0.2 × 1052.2 ± 0.3 × 10−41.9CGGC10 ± 1.02-aMeasured by competition assay (see “Experimental Procedures”).Values indicate the average and 1 S.D. of at least three experiments.2-a Measured by competition assay (see “Experimental Procedures”). Open table in a new tab Values indicate the average and 1 S.D. of at least three experiments. From these kon and koffresults, dissociation constants Kd(koff/kon) for each probe were calculated (Table II). Comparison of Kd values for AGGA and TATAMLP reveals that cisplatin-damaged DNA has about a 1.5-fold higher binding affinity to TBP compared with that of its natural binding site, the TATA box, under the same binding conditions. The Kd value (0.44 nm) for the TATA box binding to adenovirus major late promoter is consistent with previous reports (21Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar, 34Hahn S. Buratowski S. Sharp P.A. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5718-5722Crossref PubMed Scopus (219) Google Scholar, 35Parvin J.D. McCormick R.J. Sharp P.A. Fisher D.E. Nature. 1995; 373: 724-727Crossref PubMed Scopus (172) Google Scholar, 42Petri V. Hsieh M. Jamison E. Brenowitz M. Biochemistry. 1998; 37: 15842-15849Crossref PubMed Scopus (45) Google Scholar, 43Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Mol. Biol. 1998; 277: 1015-1031Crossref PubMed Scopus (61) Google Scholar, 44Wolner B.S. Gralla J.D. J. Biol. Chem. 2001; 276: 6260-6266Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 45Coleman R.A. Pugh B.F. J. Biol. Chem. 1995; 270: 13850-13859Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Previously we demonstrated that HMG domains display a distinct selectivity for platinated DNA containing different flanking sequences (26Dunham S.U. Lippard S.J. Biochemistry. 1997; 36: 11428-11436Crossref PubMed Scopus (78) Google Scholar, 46Cohen S.M. Mikata Y. He Q. Lippard S.J. Biochemistry. 2000; 39: 11771-11776Crossref PubMed Scopus (50) Google Scholar). Moreover, different flanking sequences can affect the conformational and thermodynamic properties of cisplatin-damaged oligonucleotides (47Pilch D.S. Dunham S.U. Jamieson E.R. Lippard S.J. Breslauer K.J. J. Mol. Biol. 2000; 296: 803-812Crossref PubMed Scopus (58) Google Scholar). Here, we studied the kinetics of TBP binding to cisplatin-damaged probes in diverse sequence contexts (Table I). Fig.3 shows EMSAs of TBP binding to TATAMLP and nine different cisplatin-damaged probes. Sequence selectivity is clearly manifest for TBP binding to platinated DNA. Kinetic EMSA experiments were performed to determine kon and koff values for each probe. Fig. 4 A indicates fits to EMSA data obtained from the association kinetics for six different cisplatin-damaged probes. All konvalues lie between 2.1 × 105m−1 s−1 and 1.2 × 105m−1 s−1, indicating that most of the cisplatin-damaged DNA complexes have faster association rates than that of the TATA box (Table II). The koff values were also calculated by fitting the dissociation EMSA data to Equation 2 (Fig. 4 B). The two more weakly binding probes, TGGC and CGGC, did not allow reliable kinetic data to be obtained. We therefore carried out competition assays to obtain relative Kd values for these two probes. At various concentrations of TGGC or CGGC competitor, the binding fraction of the TBP to TATAMLP was examined by EMSA and quantitated (Fig.5). From the curve fitting shown in Fig.5 B, the concentration of competitor at θ = 1/2,C1/2, was calculated. Equation 4 was employed to compute Kd values of 12 and 10 nm, respectively, for TGGC and CGGC as competitors.Figure 5Competition EMSA assay between TGGC and TATAMLP. A, a representative competition assay in which 1.4 nm TATAMLP and 2 nm TBP were mixed with increasing amounts of TGGC. B, plot of the binding fraction against the concentration of TGGC (0–600 nm).C1/2, the TGGC concentration at θ =1/2 is indicated by a dotted arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our results reveal a clear flanking sequence dependence of TBP binding to cisplatin-damaged DNA (Table II). At the N1 position, TBP prefers dA rather than T or dC, and TBP forms more stable complexes when N2 is either dA or T. A DNase I footprinting assay previously demonstrated reduced binding of TBP to the TATA box after the addition of excess cisplatin-damaged DNA (48Coin F. Frit P. Viollet B. Salles B. Egly J.M. Mol. Cell. Biol. 1998; 18: 3907-3914Crossref PubMed Scopus (45) Google Scholar). In the present study, competition EMSA experiments were performed to study the relative binding affinities of TBP to the TATA box and cisplatin-damaged DNA. TBP and the two DNA probes were mixed, and the amounts of free and bound TATA box were analyzed by using radioactively labeled TATA box DNA. Fig. 6 provides clear evidence that TBP dissociates from the TATA box upon addition of cisplatin-damaged DNA. At 1 nm TBP, more than half of the TBP-TATAMLP complex has dissociated when equivalent amounts (4 nm) of AGGA and TATAMLP are present. Upon the addition of a 3-fold excess of AGGA, most of the TBP is sequestered from the natural TATAMLP binding site. Cisplatin-modified DNA is repaired by the NER machinery. When HMG-domain proteins are added to HeLa cell-free extracts or reconstituted NER components, a significant amount of the repair of cisplatin 1,2-d(GpG) intrastrand cross-links is inhibited (39Zamble D.B" @default.
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• W2040256004 title "Kinetic Studies of the TATA-binding Protein Interaction with Cisplatin-modified DNA" @default.
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