DNA-binding Mechanism ofO 6-Alkylguanine-DNA Alkyltransferase
2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês
10.1074/jbc.m211854200
ISSN1083-351X
AutoresJoseph J. Rasimas, Anthony E. Pegg, Michael G. Fried,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O 6-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we report properties of the interaction between AGT and short DNA oligonucleotides. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This strong cooperative interaction is only slightly perturbed by active site mutation of AGT or by alkylation of either AGT or DNA. The stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged by several active site mutations or by protein or DNA alkylation. These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair. The mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O 6-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we report properties of the interaction between AGT and short DNA oligonucleotides. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This strong cooperative interaction is only slightly perturbed by active site mutation of AGT or by alkylation of either AGT or DNA. The stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged by several active site mutations or by protein or DNA alkylation. These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair. human AGT O 6-alkylguanine-DNA alkyltransferase dithiothreitol O 6-Alkylguanine-DNA alkyltransferase is a ubiquitous repair protein that plays a vital role in minimizing the mutagenic effects of alkylating agents (1Samson L. Mol. Microbiol. 1992; 6: 825-831Google Scholar, 2Sekiguchi M. Nakabeppu Y. Sakumi K. Tuzuki T. J. Cancer Res. Clin. Oncol. 1996; 122: 199-206Google Scholar, 3Pegg A.E. Dolan M.E. Moschel R.C. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 167-223Google Scholar, 4Pegg A.E. Mutat. Res. 2000; 462: 83-100Google Scholar). It catalyzes the stoichiometric transfer of a variety of alkyl substituents from theO 6-position of guanine to an active site cysteine, preventing incorrect base pairing caused by these adducts. More than 100 alkyltransferases are now known, and the crystal structures are available for three family members: the Ada-C protein from Escherichia coli (5Moore M.H. Gulbus J.M. Dodson E.J. Demple B. Moody P.C.E. EMBO J. 1994; 13: 1495-1501Google Scholar), the human alkyltransferase (hAGT)1 (6Daniels D.S. Tainer J.A. Mutat. Res. 2000; 460: 151-163Google Scholar), and the protein from the thermophilic archaeon, Pyrococcus kodakaraensis (7Hashimoto H. Inoue T. Nishioka M. Fujiwara S. Takagi M. Imanaka T. Kai Y. J. Mol. Biol. 1999; 292: 707-716Google Scholar). All of the known alkyltransferases lack the ability to dealkylate themselves, and no dealkylation activity has been found in cell extracts to date. On this basis, it is widely thought that alkyltransferase participates in a single reaction and is then irreversibly inactivated. Given the apparently nonenzymatic nature of the protein, the protection afforded by alkyltransferase is likely to depend on the regulation of its synthesis and degradation and on its ability to efficiently locate repairable lesions throughout the genome. The mechanisms by which AGT interacts with adduct-containing and adduct-free DNAs are poorly understood. Two contrasting mechanisms have been proposed to date. In the first, single AGT proteins bind normal and lesion-containing DNA, and the distribution of AGT between normal and lesion sites depends on a difference in binding affinity. This model is consistent with the observation that a single AGT monomer is necessary and sufficient to dealkylate a singleO 6-alkyl guanine adduct within a DNA duplex (3Pegg A.E. Dolan M.E. Moschel R.C. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 167-223Google Scholar). It is supported by the observation that single AGT-DNA complexes are detected by gel shift assay when AGT binds short DNA molecules. These complexes have been interpreted as having a 1:1 AGT:DNA stoichiometry, and the binding affinities have been calculated based on that assumption (8Bender K.B. Federwisch M. Loggen U. Nehls P. Rajewsky M.F. Nucleic Acids Res. 1996; 24: 2087-2094Google Scholar). The second mechanism was proposed when it was found that some AGT-DNA complexes have stoichiometries greater than 1:1 and form without the accumulation of detectable binding intermediates (9Fried M.G. Kanugula S. Bromberg J.L. Pegg A.E. Biochemistry. 1996; 35: 15295-15301Google Scholar). This pattern strongly suggests a cooperative binding mechanism for AGT. Here we more thoroughly characterize the cooperative binding mechanism. We show that it functions on both single-stranded and duplex 16-mer DNAs and with unmodified and alkylated hAGTs. In all cases the stoichiometries of hAGT:16-mer complexes were 4:1. In this binding mode, hAGT discriminates poorly between lesion-containing and lesion-free DNA. Together these results support a novel model of binding site search and recognition that involves the cooperative formation and processive movement of multi-protein complexes. T4 polynucleotide kinase was purchased from New England Biolabs, and [γ-32P]ATP was purchased from PerkinElmer Life Sciences. Acrylamide andN,N′-methylene bisacrylamide were purchased from Aldrich. O 6-Methylguanine andO 6-benzylguanine were generously provided by Dr. R. C. Moschel (ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD). Recombinant human AGT (wild type and C145A mutant proteins) were prepared as previously described (10Pegg A.E. Boosalis M. Samson L. Moschel R.C. Byers T.L. Swenn K. Dolan M.E. Biochemistry. 1993; 32: 11998-12006Google Scholar). Both of the proteins were homogeneous as judged by electrophoresis (not shown). The wild type protein was 100% active in debenzoylatingO 6-benzylguanine. The C145A mutant lacks the active site cysteine and is not active against alkyl-DNA or alkyl-guanine substrates (11Crone T.M. Pegg A.E. Cancer Res. 1993; 53: 4750-4753Google Scholar). The construction of modified pQE-30 vectors encoding C-terminally His6-tagged wild type and C145A and C145S mutant AGT proteins for expression in E. coli was accomplished as described (12Liu H. Xu-Welliver M. Pegg A.E. Mutat. Res. 2000; 452: 1-10Google Scholar). His-tagged proteins were purified from cell lysates using TALON® affinity resin (Clontech), according to the manufacturer's instructions. The samples were dialyzed against 50 mm Tris buffer (pH 7.6) containing 5 mm DTT and stored frozen at −80 °C until needed. Wild type hAGT proteins were alkylated at the active site cysteine by incubation (at 37 °C for 30 min) with either 1 mm O 6-methylguanine or 1 mm O 6-benzylguanine according to Kanugula et al. (13Kanugula S. Goodtzova K. Pegg A.E. Biochem. J. 1998; 329: 545-550Google Scholar). Alkylation was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of trypsin-digested protein, in which the conversion of the fragment containing the active site cysteine (Gly136–Arg147,M predicted = 1314.72,m/z obs = 1315.82) to its methylated derivative (M predicted = 1328.73,m/z obs = 1328.81) or benzoylated derivative (M predicted = 1391.80,m/z obs = 1393.66) was observed. Complete conversion of hAGT to the alkyl form (as monitored by mass spectrometry) eliminated detectable alkyl transferase activity assayed with [3H]methyl calf thymus DNA as substrate (results not shown) (14Xu-Welliver M. Kanugula S. Pegg A.E. Cancer Res. 1998; 58: 1936-1945Google Scholar). Human AGT undergoes a conformational change upon alkylation that reduces its in vivo and in vitrohalf-life (13Kanugula S. Goodtzova K. Pegg A.E. Biochem. J. 1998; 329: 545-550Google Scholar). Accordingly, the samples were used immediately following alkylation to avoid problems of instability. Human AGT concentrations were measured both with the BCA dye binding assay (15Walker J.M. Methods Mol. Biol. 1994; 32: 5-8Google Scholar) and spectrophotometrically using a molar extinction coefficient, ε280 = 3.93 × 104m−1 cm−1, calculated from data of Roy et al. (16Roy R. Shiota S. Kennel S.J. Raha R. von Wronski M. Brent T.P. Mitra S. Carcinogenesis. 1995; 16: 405-411Google Scholar). The values of ε215/ε280 = 8.2 and ε260/ε280 = 0.63 were obtained from UV spectra of the purified protein dissolved in 10 mm Tris buffer (pH 7.6) at 21 °C. A 16-residue oligodeoxyribonucleotide (sequence 5′-GAC TGA CTG ACT GAC T-3′) and its complement were purchased from Invitrogen. A substrate oligonucleotide with the same sequence and a methyl substitution at theO 6-position of the 3′-most guanine (shown above in bold type) was purchased from Synthegen LLC (Houston, TX). When duplex DNA was required, the oligonucleotide samples were combined and annealed as described (17Kanugula S. Goodtzova K. Edara S. Pegg A.E. Biochemistry. 1995; 34: 7113-7119Google Scholar). The DNA samples were labeled at the 5′ termini with 32P as described by Maxam and Gilbert (18Maxam A.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 560-565Google Scholar) and transferred into 10 mm Tris (pH 8.0 at 21 °C) using Sephadex G-25 centrifuge columns (Amersham Biosciences). Stock DNA concentrations were measured spectrophotometrically, using ε260 = 1.3 × 104m−1 cm−1 (per base pair) for duplex samples and ε260 = 1.04 × 104m−1 cm−1 (per base) for single-stranded samples. The binding reactions were carried out at 20 ± 1 °C in 10 mm Tris (pH 7.6), 1 mm dithiothreitol, and 10 μg/ml bovine serum albumin, supplemented with NaCl as indicated. Protein-DNA complexes were formed by adding appropriate amounts of hAGT to solutions containing 32P-labeled oligodeoxyribonucleotides. The mixtures were equilibrated at 20 ± 1 °C for 30 min. Duplicate samples incubated for longer periods gave identical results, indicating that equilibrium had been attained. Electrophoresis was performed using 10% polyacrylamide gels (acrylamide:N,N′-methylene bisacrylamide = 75:1), cast, and run at 8 V/cm in buffer consisting of 10 mm Tris acetate (pH 7.6) supplemented with NaCl to match the conductivity of the protein-DNA samples. Autoradiograms were obtained with Kodak X-Omat Blue XB-1 film exposed at 4 °C. Gel segments containing individual electrophoretic species were excised using the developed film as a guide and counted in a scintillation counter by the Cerenkov method (20van Holde K.E. Physical Biochemistry. Prentice Hall, Englewood Cliffs, NJ1985: 51-92Google Scholar). Similar results were obtained using scanning densitometry. The serial dilution method (19Fried M.G. Crothers D.M. J. Mol. Biol. 1984; 172: 241-262Google Scholar) was used to obtain self-consistent estimates of the binding stoichiometry (n) and the association constant (K a). For a binding mechanism of the type nP + D ⇄P n D, the association constant isK n = [PnD]/[P]n[D]. Separating variables and taking logarithms gives the following equation.ln[PnD][D]=nln[P]+lnKnEquation 1 Dilution of an AGT-DNA mixture changes the binding ratio [PnD]/[D] by mass action. A recursive method (14Xu-Welliver M. Kanugula S. Pegg A.E. Cancer Res. 1998; 58: 1936-1945Google Scholar) was used to evaluate n and K a, starting with an initial value of n = 4 deduced from the value ofM r (complex) measured by sedimentation equilibrium. In many cases the association constant was also evaluated by direct titration. hAGT protein was directly added to 32P-DNA solutions (typically ∼5 × 10−7m), and the samples were analyzed by native gel electrophoresis. The free protein concentration [P] was estimated using the conservation relation [P] = [P]o − n[PnD] in which [P]o is the total hAGT concentration in the reaction mixture, and an initial value of n = 4 was assumed on the basis of our sedimentation equilibrium results (see Fig.1). For the highly cooperative formation of a 4:1 complex under conditions of large protein excess, the fractional saturationY is given by (20van Holde K.E. Physical Biochemistry. Prentice Hall, Englewood Cliffs, NJ1985: 51-92Google Scholar) the following.Y=[P4D][D]+[P4D]=[P]41/Ka+[P]4Equation 2 Estimates of K a were obtained by fitting Equation 2 to the experimentally determined dependence of Yon [P]. hAGT protein and oligodeoxyribonucleotides were dialyzed against 10 mm Tris (pH 7.6), 1 mm DTT, 1 mm EDTA, 100 mm NaCl. Analytical ultracentrifugation was performed at 20 ± 0.1 °C in a Beckman XL-A centrifuge using an AN 60 Ti rotor. Scans were obtained at 260 and 280 nm with a step size of 0.001 cm. Equilibrium was considered to be attained when scans made 6 h apart were indistinguishable. Typically, equilibration times ≥24 h met this criterion for AGT-DNA mixtures. Five scans were averaged for each sample at each wavelength and rotor speed. For analysis of hAGT protein alone, models incorporating different assembly stoichiometries were based on the following general equation. A(r)=∑nαnexp[σn(r2−ro2)]+εEquation 3 Here A(r) is the absorbance at radial positionr, and αn is the absorbance of the nth species at the reference radius (r o). The parameter ςn is the reduced molecular weight [ςn =M n(1 −v̄ρ)ω2/(2RT)],M n is the molecular weight of the nth species, v̄ its partial specific volume, ρ is the solvent density, ω is the rotor angular velocity, R is the gas constant, T is the absolute temperature, and ε is the base-line offset. Solvent density (1.004 g/ml) was measured using a Mettler density meter. The partial specific volume of hAGT (0.744 ml/g) was calculated by the method of Cohn and Edsall (21Cohn E.J. Edsall J.T. Proteins, Amino Acids and Peptides as Ions and Dipolar Ions. Reinhold, New York1943: 140-154Google Scholar), using partial specific volumes of amino acids tabulated by Laue et al.(22Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Harding J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. The Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar). For a system in which hAGT protein is in binding equilibrium with DNA according to the mechanism nP + D ⇄P n D, Equation 3 becomesA(r)=αPexp[σP(r2−ro2)]+αDexp[σD(r2−ro3)]+ αPnDexp[σPnD(r2−ro2)]+εEquation 4 Here, most terms are defined as for Equation 3; αDand αPnD are absorbances of DNA and protein-DNA complex at r o, the reduced molecular weights of DNA and protein-DNA complex are given by ςD =M D(1 −v̄ Dρ)ω2/(2RT) and ςPnD = (nM P +M D)(1 −v̄ PnDρ)ω2/(2RT), and n is the protein:DNA ratio of the complex. In this analysis, the known molecular weights of recombinant hAGT proteins (21,614 ≤ M r ≤ 21,876) and DNA (M r = 4,881 for single-stranded DNA andM r = 9,762 for double-stranded DNA) were used as constants. The partial specific volume of NaDNA at 0.1 mNaCl (0.502 ml/g) was estimated by interpolation of the data of Cohen and Eisenberg (23Cohen G. Eisenberg H. Biopolymers. 1968; 6: 1077-1100Google Scholar). Partial specific volumes of each of protein-DNA complexes were estimated using Equationv¯PnD=(nMPu¯P+MDu¯D)(nMP+MD)Equation 5 Here n is the stoichiometric ratio of protein to DNA in the complex. Equation 5 is based on the assumption that there is no significant change in partial specific volumes of the components upon association. Although we do not know whether such a volume change occurs, it seems reasonable that values ofv̄ PnD for complexes containing a large mass proportion of protein (like those analyzed here) should reflect that proportion. Equation 4 was used in global analysis of multiple data sets obtained at different macromolecular concentrations and/or rotor speeds (24Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Google Scholar). In this method, the values of αP, αD, αPnD, and ε are unique to each sample, but the value of n must be common to all of the data sets. Nonideality was not considered, because there was no evidence of nonideal effects. Solutions containing hAGT protein at two nominal concentrations (3.6 and 13.7 μm) were brought to sedimentation equilibrium at three different centrifuge speeds (22,000, 31,000, and 43,000 rpm). A representative data set for a 13.7 μm sample of His6-tagged C145S hAGT taken at 43,000 rpm and 20 °C is shown in Fig.1 (curve A). The solid line is the result of fitting the single-species version of Equation 3 (n = 1) to the data. This fit returned a value of M r = 21,800 ± 400, which is in excellent agreement with the monomer molecular weight derived from the protein sequence (M r = 21,860). The small, uniformly distributed residuals indicate that the monomer model is consistent with the data over the entire concentration range present in the centrifuge cell. Extension of the model to include oligomers of hAGT (Equation 3 with n > 1 and M n= nM1) did not improve the quality of the fit as judged by the correlation coefficient or by the magnitude of the residuals (result not shown). Similar results were obtained for wild type hAGT (M r = 21,500 ± 200), C145A hAGT (M r = 21,400 ± 200), His6-tagged wild type hAGT (M r = 22,000 ± 500), and His6-tagged C145A hAGT (M r = 21,800 ± 200). These molecular weights agree well with values predicted from protein sequence, consistent with the interpretation that all of the preparations were monomeric within the concentration range tested. Importantly, neither the His6 tag nor the active site mutation C145A changed the monomeric state of free hAGT. As discussed below, hAGT forms oligomeric complexes with DNA. The absence of detectable hAGT oligomers in the absence of DNA demonstrated here argues against models in which protein association precedes DNA binding. Mixtures containing hAGT and single-stranded DNA were brought to sedimentation equilibrium at four different centrifuge speeds (11,000, 15,000, 20,500, and 27,000 rpm). Representative data are shown in Fig. 1(curve B); the solid curve represents the global fit of Equation 4 to the data ensemble. The small, uniformly distributed residuals indicate that the simple mechanism nP+ D ⇄ P n D, withn = 3.98 ± 0.07 is consistent with the data. Sedimentation models with additional species did not fit the data significantly better than Equation 4 (results not shown). This outcome is intriguing because it suggests that then = 4 complex forms without significant accumulation of intermediates of lower stoichiometry. This interpretation is supported by the gel mobility shift experiments described below. Parallel experiments carried out with wild type and C145A, His6wild type, His6-C145A, and His6-C145S hAGTs returned closely similar protein-DNA stoichiometries (TableI), indicating that neither modification of the active site cysteine nor presence of a C-terminal His6 tag alters the stoichiometry of association.Table IAssociation constants and binding stoichiometries for the interaction of 16-mer oligodeoxyribonucleotides with recombinant hAGT proteinsProteinSingle-stranded 16-mer DNADouble-stranded 16-mer DNAStoichiometryK a/1024m−4K mono/(105m−1)aFormation constants for the 4:1 AGT-DNA complex are in units of 1024m−4. The values given in parentheses are monomer-equivalent association constants (units of 105m−1).StoichiometryK a/1024m−4K mono/(105m−1)aFormation constants for the 4:1 AGT-DNA complex are in units of 1024m−4. The values given in parentheses are monomer-equivalent association constants (units of 105m−1).Wild type hAGT3.89 ± 0.2bStoichiometry determined by serial dilution method.0.145 ± 0.024(6.15 ± 0.28)eAssociation constant determined by serial dilution method.3.93 ± 0.2bStoichiometry determined by serial dilution method.0.836 ± 0.093(9.56 ± 0.27)eAssociation constant determined by serial dilution method.3.90 ± 0.4cStoichiometry determined by continuous variation analysis.0.154 ± 0.019(6.26 ± 0.20)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.4.04 ± 0.3cStoichiometry determined by continuous variation analysis.0.842 ± 0.16(9.55 ± 0.46)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.4.02 ± 0.10dStoichiometry determined by sedimentation equilibrium.3.89 ± 0.13dStoichiometry determined by sedimentation equilibrium.C145A hAGT3.92 ± 0.2bStoichiometry determined by serial dilution method.0.125 ± 0.012(5.96 ± 0.21)eAssociation constant determined by serial dilution method.3.96 ± 0.1bStoichiometry determined by serial dilution method.0.434 ± 0.086(8.12 ± 0.40)eAssociation constant determined by serial dilution method.3.94 ± 0.3cStoichiometry determined by continuous variation analysis.0.121 ± 0.014(5.89 ± 0.17)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.3.89 ± 0.5cStoichiometry determined by continuous variation analysis.0.427 ± 0.11(8.03 ± 0.51)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.His6-Wild type hAGT3.87 ± 0.2bStoichiometry determined by serial dilution method.0.300 ± 0.079(7.39 ± 0.52)eAssociation constant determined by serial dilution method.3.85 ± 0.2bStoichiometry determined by serial dilution method.23.0 ± 4.1(21.9 ± 0.98)eAssociation constant determined by serial dilution method.3.89 ± 0.2cStoichiometry determined by continuous variation analysis.0.305 ± 0.045(7.42 ± 0.28)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.3.90 ± 0.3cStoichiometry determined by continuous variation analysis.27.5 ± 12(22.5 ± 2.5)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.3.99 ± 0.06dStoichiometry determined by sedimentation equilibrium.4.07 ± 0.11dStoichiometry determined by sedimentation equilibrium.His6-C145A hAGT3.92 ± 0.1bStoichiometry determined by serial dilution method.0.269 ± 0.059(7.20 ± 0.39)eAssociation constant determined by serial dilution method.3.88 ± 0.2bStoichiometry determined by serial dilution method.13.4 ± 7.5(19.1 ± 2.8)eAssociation constant determined by serial dilution method.3.90 ± 0.2cStoichiometry determined by continuous variation analysis.0.274 ± 0.076(7.18 ± 0.51)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.3.96 ± 0.3cStoichiometry determined by continuous variation analysis.15.0 ± 9.4(18.8 ± 3.4)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.His6-C145S hAGT3.84 ± 0.2bStoichiometry determined by serial dilution method.0.688 ± 0.098(9.16 ± 0.53)eAssociation constant determined by serial dilution method.3.99 ± 0.1bStoichiometry determined by serial dilution method.121 ± 14(33.1 ± 0.69)eAssociation constant determined by serial dilution method.3.88 ± 0.4cStoichiometry determined by continuous variation analysis.0.697 ± 0.013(9.11 ± 0.41)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.4.12 ± 0.3cStoichiometry determined by continuous variation analysis.161 ± 95(34.3 ± 5.7)fAssociation constant determined by direct titration method, assuming a binding stoichiometry.3.98 ± 0.07dStoichiometry determined by sedimentation equilibrium.3.94 ± 0.08dStoichiometry determined by sedimentation equilibrium.a Formation constants for the 4:1 AGT-DNA complex are in units of 1024m−4. The values given in parentheses are monomer-equivalent association constants (units of 105m−1).b Stoichiometry determined by serial dilution method.c Stoichiometry determined by continuous variation analysis.d Stoichiometry determined by sedimentation equilibrium.e Association constant determined by serial dilution method.f Association constant determined by direct titration method, assuming a binding stoichiometry. Open table in a new tab Similar experiments were carried out with hAGT and a 16-bp duplex DNA. Samples were brought to sedimentation equilibrium at 11,000, 15,000, 20,500, and 27,000 rpm. Representative data are shown in Fig. 1(curve C); the solid curve represents the global fit of Equation 4 to the data ensemble. As before, the high quality of the fit indicates that the simple mechanism nP +D ⇄ P n D, withn = 3.94 ± 0.08 is consistent with the data. Inclusion of additional species in the sedimentation model did not improve the quality of the fit (results not shown), suggesting that, as with single-stranded DNA, stoichiometric intermediates are not present in significant concentrations. This suggestion is supported by gel shift results (discussed below). The fact that the hAGT stoichiometry is the same, within error, for both single-stranded and duplex 16-mers is intriguing, because it suggests that factors determining stoichiometry may not be sensitive to the association state of the DNA. Electrophoretic mobility shift assays (25Fried M.G. Crothers D.M. Nucleic Acids Res. 1981; 9: 6505-6525Google Scholar) were performed to explore a range of hAGT and DNA concentrations below those accessible in the analytical ultracentrifuge. The binding of hAGT to DNA produced a single mobility-shifted complex at all protein and DNA concentrations that gave detectable binding (Fig.2 A). This binding pattern is consistent with a mechanism of the type nP + D⇄ P n D in which the maximum stoichiometry (n) is reached without accumulation of significant concentrations of intermediates (19Fried M.G. Crothers D.M. J. Mol. Biol. 1984; 172: 241-262Google Scholar). The dependence of ln([PD]/[D]) on ln[P] is shown in Fig. 2 B. The values of stoichiometry and K a were calculated from these data as described under "Experimental Procedures." The stoichiometry values most consistent with the data for His6-tagged C145S hAGT binding to 16-mer are 3.84 ± 0.2 for single-stranded DNA and 3.99 ± 0.1 for double-stranded DNA. These stoichiometry values (summarized in Table I) agree well with ones obtained by analytical ultracentrifugation, despite a difference in the salt concentration of the buffers used in the two techniques (serial dilution assays, ∼10 mm; sedimentation equilibrium assays, ∼110 mm). Closely similar stoichiometry values were obtained by the electrophoretic mobility shift assay method for the active site mutant C145A hAGT as well as His6-wild type hAGT and His6-C145A hAGT (TableI), supporting the conclusion that neither the C145A modification of the active site nor the presence of a C-terminal His6 tag has a significant effect on the stoichiometry of these interactions. Binding stoichiometries were also measured by the continuous variation (Job plot) method (26Huang C.Y. Methods Enzymol. 1982; 87: 509-525Google Scholar). With the input concentrations of protein and DNA ([P]o + [D]o) held constant, the ratio of [P]o:[D]o that yields the greatest concentration of complex (the optimal combining ratio) is a measure of the association stoichiometry. As shown in Fig.3 and summarized in Table I, this method returns hAGT-DNA stoichiometries close to 4:1, in good agreement with values obtained by the serial dilution and analytical ultracentrifuge methods. Taken with the fact that higher stoichiometry complexes are readily observed with larger DNAs (9Fried M.G. Kanugula S. Bromberg J.L. Pegg A.E. Biochemistry. 1996; 35: 15295-15301Google Scholar), 2J. J. Rasimas, unpublished results. the absence of detectable complexes with stoichiometries greater than 4:1, even at high [hAGT], suggests that this stoichiometry represents protein saturation for both single-stranded and duplex 16-mer DNAs. Together, the presence of free DNA in equilibrium with the 4:1 complex and the absence of complexes with protein:DNA ratios <4:1, suggest that hAGT binds cooperatively to both single-stranded and double-stranded DNAs. Because free hAGT is monomeric (Fig. 1), this pattern suggests that the protein assembly forms on DNA and not in free solution prior to DNA binding. Association constants for the interaction of wild type and C145A, His6-wild type, His6 C145A, and His6-C145S hAGTs with single-stranded and duplex 16-mer DNAs were calculated from serial dilution data as described above and were also determined by direct titration of DNA by hAGT (Fig. 4; data summarized in Table I). Measured in these ways, the formation constant (K a) for the complex of wild type protein with single-stranded 16-mer is ∼1.5 × 1023m−4. Assuming equipartition of the binding free energies among the four hAGT monomers, this corresponds to a monomer association constant of ∼6.2 × 105m−1, which is in reasonable agreement with values reported for small (9Fried M.G. Kanugula S. Bromberg J.L. Pegg A.E. Biochemistry. 1996; 35: 15295-15301Google Scholar) and large (27Chan C.Z.W. Ciardelli T. Eastman A. Bresnick E. Arch. Biochem. Biophys. 1993; 300: 193-200Google Scholar) DNA molecules. The very narrow range of association constants for wild type, active site mutant, and His6-tagged hAGT proteins (best seen by comparison of the effective monomer association constants given in Table II) is an especially interesting result. It indicates that neither the C145A mutation of the active site nor the presence of a C-terminal His6 affinity tag signi
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