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• W2007411488 abstract "A combination of hydrodynamic and cross-linking studies were used to investigate self-assembly of the Escherichia coli DNA repair protein UvrB. Though the procession of steps leading to incision of DNA at sites flanking damage requires that UvrB engage in an ordered series of complexes, successively with UvrA, DNA, and UvrC, the potential for self-association had not yet been reported. Gel permeation chromatography, nondenaturing polyacrylamide gel electrophoresis, and chemical cross-linking results combine to show that UvrB stably assembles as a dimer in solution at concentrations in the low micromolar range. Smaller populations of higher order oligomeric species are also observed. Unlike the dimerization of UvrA, an initial step promoted by ATP binding, the monomer-dimer equilibrium for UvrB is unaffected by the presence of ATP. The insensitivity of cross-linking efficiency to a 10-fold variation in salt concentration further suggests that UvrB self-assembly is driven largely by hydrophobic interactions. Self-assembly is significantly weakened by proteolytic removal of the carboxyl terminus of the protein (generating UvrB*), a domain also known to be required for the interaction with UvrC leading to the initial incision of damaged DNA. This suggests that the C terminus may be a multifunctional binding domain, with specificity regulated by protein conformation. A combination of hydrodynamic and cross-linking studies were used to investigate self-assembly of the Escherichia coli DNA repair protein UvrB. Though the procession of steps leading to incision of DNA at sites flanking damage requires that UvrB engage in an ordered series of complexes, successively with UvrA, DNA, and UvrC, the potential for self-association had not yet been reported. Gel permeation chromatography, nondenaturing polyacrylamide gel electrophoresis, and chemical cross-linking results combine to show that UvrB stably assembles as a dimer in solution at concentrations in the low micromolar range. Smaller populations of higher order oligomeric species are also observed. Unlike the dimerization of UvrA, an initial step promoted by ATP binding, the monomer-dimer equilibrium for UvrB is unaffected by the presence of ATP. The insensitivity of cross-linking efficiency to a 10-fold variation in salt concentration further suggests that UvrB self-assembly is driven largely by hydrophobic interactions. Self-assembly is significantly weakened by proteolytic removal of the carboxyl terminus of the protein (generating UvrB*), a domain also known to be required for the interaction with UvrC leading to the initial incision of damaged DNA. This suggests that the C terminus may be a multifunctional binding domain, with specificity regulated by protein conformation. In the characterization of any protein, fundamental questions include whether the monomeric structure is complete, and the identity of other macromolecules with which it can functionally interact. The answers to these take on added importance for the components of the nucleotide excision repair pathway (NER) 1The abbreviations used are:NERnucleotide excision repairPAGEpolyacrylamide gel electrophoresisDTTdithiothreitolMOPS4-morpholinepropanesulfonic acidMe2SOdimethyl sulfoxideDMSdimethylsuberimidateDSGdisuccinimidyl glutarate in Escherichia coli. Introduction of dual incisions surrounding a damage site in DNA by the NER proteins UvrA, UvrB, and UvrC, requires an ordered succession of association/dissociation steps, mediated at multiple points along the pathway by nucleotide binding and/or catalysis. As detailed in recent reviews (1Grossman L. Thiagalingam S. J. Biol. Chem. 1993; 268: 16871-16874Abstract Full Text PDF PubMed Google Scholar, 2Van Houten B. Microbiol. Rev. 1990; 54: 18-51Crossref PubMed Google Scholar, 3Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (965) Google Scholar), the following sequence of steps have been suggested, focusing here on the shifting identity and stoichiometry of the macromolecular components of the repair complex. nucleotide excision repair polyacrylamide gel electrophoresis dithiothreitol 4-morpholinepropanesulfonic acid dimethyl sulfoxide dimethylsuberimidate disuccinimidyl glutarate Dimerization of the UvrA protein, promoted by ATP binding, enables formation of a protein-DNA complex, initially at an undamaged site (4Oh E.Y. Claassen L. Thiagalingam S. Mazur S. Grossman L. Nucleic Acids Res. 1989; 17: 4145-4159Crossref PubMed Scopus (60) Google Scholar,5Mazur S.J. Grossman L. Biochemistry. 1991; 30: 4432-4443Crossref PubMed Scopus (84) Google Scholar). Recruitment of UvrB, which does not on its own bind DNA, generates a functional UvrA2-UvrB1-DNA complex, with this stoichiometry assumed from that demonstrated for an ATP-dependent UvrA-UvrB interaction (DNA not present) in solution (6Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Crossref PubMed Scopus (167) Google Scholar). At this stage, the repair proteins can disengage from the initial DNA binding site, allowing translocation or delivery to the DNA lesion (7Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar). Damage recognition is marked by formation of a stable protein-DNA complex (8Yeung A.T. Mattes W.B. Grossman L. Nucleic Acids Res. 1986; 14: 2567-2582Crossref PubMed Scopus (41) Google Scholar), from which UvrA dissociates (6Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Crossref PubMed Scopus (167) Google Scholar, 9Bertrand-Burggraf E. Selby C.P. Hearst J.E. Sancar A. J. Mol. Biol. 1991; 219: 27-36Crossref PubMed Scopus (54) Google Scholar, 10Visse R. de Ruijter M. Moolenaar G.F. van de Putte P. J. Biol. Chem. 1992; 267: 6736-6742Abstract Full Text PDF PubMed Google Scholar), leaving a UvrB-DNA pre-incision complex. The DNA in this complex is acutely kinked (11Shi Q. Thresher R. Sancar A. Griffith J. J. Mol. Biol. 1992; 226: 425-432Crossref PubMed Scopus (78) Google Scholar), a structural change necessary for later incision and either facilitated by or stabilized by contacts with UvrB (12Hsu D.S. Kim S.-T. Sun Q. Sancar A. J. Biol. Chem. 1995; 270: 8319-8327Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). It is likely that conformational change(s) also occur in the UvrB protein to augment its affinity for DNA; in solution, nucleotide binding by UvrB has been shown to be limited to short oligonucleotides bearing damage (12Hsu D.S. Kim S.-T. Sun Q. Sancar A. J. Biol. Chem. 1995; 270: 8319-8327Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Addition of UvrC is enabled by conformational changes in the UvrB-damaged DNA pre-incision complex (13Delagoutte E. Bertrand-Burggraf E. Dunand J. Fuchs R.P.P. J. Mol. Biol. 1997; 266: 703-710Crossref PubMed Scopus (23) Google Scholar) and prompts a further conformational change that is dependent in rate on the nature of damage (14Visse R. van Gool A.J. Moolenaar G.F. de Ruijter M. van de Putte P. Biochemistry. 1994; 33: 1804-1811Crossref PubMed Scopus (31) Google Scholar). These steps complete the endonucleolytic-competent complex that finally cleaves the DNA backbone in the hallmark pattern of NER, first four to five residues 3′, and then eight phosphodiester bonds 5′ to the lesion (15Sancar A. Rupp W.D. Cell. 1983; 33: 249-260Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 16Yeung A.T. Mattes W.B. Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6157-6161Crossref PubMed Scopus (95) Google Scholar, 17Visse R. de Ruijter M. Brouwer J. Brandsma J.A. van de Putte P. J. Biol. Chem. 1991; 266: 7609-7617Abstract Full Text PDF PubMed Google Scholar, 18Lin J.J. Phillips A.M. Hearst J.E. Sancar A. J. Biol. Chem. 1992; 267: 17693-17700Abstract Full Text PDF PubMed Google Scholar, 19Zou Y. Liu T.-M. Geacintov N.E. Van Houten B. Biochemistry. 1995; 34: 13582-13593Crossref PubMed Scopus (77) Google Scholar). As noted with DNA, UvrB has no or an extremely weak affinity for UvrC when the two are simply introduced in solution. The addition of UvrC to the repair complex is dependent both on the presence of the C-terminal domain of UvrB, and a conformational change that apparently renders this interaction surface accessible (20Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). As a nexus in the progression of pre-incision steps, the malleable UvrB is thus called upon to display, at the appropriate times, binding affinities that are masked in the isolated protein. The catalytic properties of UvrB, as well as its associative properties, are markedly altered along the repair pathway. In particular, expression of the DNA-dependent ATPase of UvrB, an activity that is cryptic in the isolated protein, has been shown to be essential for release of the A2-B-DNA from the initial binding site (7Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar), in formation of the pre-incision complex (21Moolenaar G.F. Visse R. Ortiz-Buysse M. Goosen N. van de Putte P. J. Mol. Biol. 1994; 240: 294-307Crossref PubMed Scopus (67) Google Scholar), in supporting the 5′ incision by the UvrB-UvrC nuclease (22Zou Y. Walker R. Bassett H. Geacintov N.E. van Houten B. J. Biol. Chem. 1997; 272: 4820-4827Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and likely in the translocation of the repair proteins to the damage site (evidence reviewed in Ref. 1Grossman L. Thiagalingam S. J. Biol. Chem. 1993; 268: 16871-16874Abstract Full Text PDF PubMed Google Scholar). Little is known in detail of the manifold protein-protein and protein-DNA interactions at any given point along the pathway and less of the regulation of transitions between associations, structurally or kinetically. Whether step-specific changes in stoichiometry may occur, as has been demonstrated qualitatively for subunit composition (e.g. dissociation of UvrA before recruitment of UvrC), has not yet been addressed. A more complete understanding of the potential for interactions of any of the repair proteins could only aid in our understanding of this complex mechanism by which damage of a remarkably broad spectrum is recognized and removed. In our purification of UvrB, we observed that its elution volume from a gel exclusion column, compared with that of protein standards, suggested a hydrodynamic volume approximately twice that predicted from the molecular weight of UvrB. This led us to broaden the scope of an earlier hydrodynamic study so as to re-examine the generality of the reported finding (6Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Crossref PubMed Scopus (167) Google Scholar) that UvrB exists solely as a monomer, and whether one may then infer that UvrB-UvrB subunit interactions could play no role in complex assembly. We present evidence here, derived from both hydrodynamic and chemical cross-linking techniques, that self-association, the formation of dimers and possibly higher order oligomers, is within the panoply of UvrB interactions and is likely to occur under physiological conditions. We also show that truncation of the protein by theompT protease, which yields a product termed UvrB*, significantly impairs self-association. UvrB* is of interest in (at least) two ways. The residues eliminated are at the C terminus, and have been shown to be required for recruitment of UvrC to the incision complex (20Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and because the cryptic DNA-dependent ATPase of UvrB is active in UvrB*, offering the possibility that it may serve as a model for conformational changes induced normally by interaction with UvrA. The source, purification, and quantitation of UvrB protein have recently been described in detail (23Hildebrand E.L. Grossman L. J. Biol. Chem. 1998; 273: 7818-7827Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Briefly, theuvrB gene, under the regulation of its own promoters, was originally cloned into a pTZ19R plasmid from the E. colistrain AB1157 and expressed in N364 (W3110 gal+, sup0, F−, Δ(attB-bio-uvrB)), auvrB deletion strain of E. coli K-12 (obtained from M. Gottesman, Columbia University). Specific proteolysis of the UvrB proteins, using the ompT expression system UT5600/pmL19 (24Grodberg J. Dunn J.J. J. Bacteriol. 1988; 170: 1245-1253Crossref PubMed Scopus (576) Google Scholar), and purification of the UvrB* product also followed published protocol (7Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar). The hydrodynamic radius and by implication the assembly state of UvrB and its potential dependence on initial protein concentration was examined using a 1.0 × 30 cm (10/30) Superose 12 HR column, operated at room temperature (22–24 °C) by a fast protein liquid chromatography system (Amersham Pharmacia Biotech). The buffer used with this column was 50 mm HEPES, pH 7.6, 300 mm KCl, 10 mm MgCl2, 5% glycerol, 2 mm DTT. Glycerol content of the buffer had been reduced to keep operating pressure lower; this ranged from approximately 0.75–1.0 megapascals during sample and standard runs at a flow rate of 0.3 ml/min. Sample was loaded in a volume of 0.1 ml, and elution from the column was monitored by flow-through absorbance measurements at 280 nm, with 0.3 ml fractions collected as a check on elution volume and protein integrity (as judged by SDS-PAGE). Calibration standards were purchased from Sigma (MW-GF-200 kit, supplemented with apoferritin and thyroglobulin to extend the molecular weight range to 669,000), and included blue dextran (M r 2,000,000) for void volume determination. The partition coefficient,K av = (V e −V 0)/(V t −V 0), where V e is the elution volume, V 0, the void volume, andV t, the total gel volume, was calculated for standards and samples from triplicate runs (25Laurent T.C. Killander J. J. Chromatogr. 1964; 14: 317-330Crossref Google Scholar, 26Ackers G.K. Neurath H. Hill R.L. The Proteins. 1. Academic Press, New York1975: 1-94Crossref Google Scholar). The Stokes radii values for standards were taken from the literature (27Potschka M. Anal. Biochem. 1987; 162: 47-64Crossref PubMed Scopus (122) Google Scholar), except for β-amylase, for which a value of 5.18 nm was calculated from the published sedimentation coefficient and ancillary data (28Spradlin J. Thoma J.A. J. Biol. Chem. 1970; 245: 117-127Abstract Full Text PDF PubMed Google Scholar). Oligomers of UvrB were resolved on a 4–30% polyacrylamide gradient gel (375 mm Tris·Cl, pH 8.8); a 3% stacking gel (125 mm Tris·Cl, pH 6.8) was used to further improve resolution. The denaturant SDS was omitted from the gels and from both electrophoresis (25 mm Tris·Cl, 250 mm glycine, pH 8.3) and sample buffers (which contained a final concentration of 10% glycerol and 0.05% bromphenol blue). Electrophoresis was performed at 100 V to 2,000 V-h at room temperature, or at 150 V to 3,000 V-h at 4 °C. Protein bands were visualized by Coomassie Brilliant Blue R250 staining. Molecular weight standards were obtained from Amersham Pharmacia Biotech (HMW kit). The bifunctional imidoester dimethyl suberimidate (DMS) (29Davies G.E. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 651-656Crossref PubMed Scopus (719) Google Scholar) is reactive primarily with lysine residues, and has long been used as a protein cross-linking reagent (30Peters K. Richards F.M. Annu. Rev. Biochem. 1977; 46: 523-551Crossref PubMed Scopus (445) Google Scholar). UvrB and UvrB* proteins, at 5 μm, were allowed to equilibrate for 10 min at room temperature in a buffer of 20 mm HEPES, pH 7.6, 10% glycerol, 1 mm DTT and KCl at 25, 100, or 300 mm, and ± ATP to 8 mm, before addition of cross-linker, bringing the reaction volume to 20 μl. DMS (Fluka BioChemika) was then added to a final concentration of 5 mg/ml from a freshly prepared 25 mg/ml stock solution in 0.5 mtriethanolamine buffer, pH 8.5 (31Chao K. Lohman T.M. J. Mol. Biol. 1991; 221: 1165-1181Crossref PubMed Scopus (84) Google Scholar). An additional 1-μl aliquot of the DMS stock (an increment of 1.2 mg/ml) was added after 15 min of incubation, owing to the short lifetime of this reagent in aqueous solution. After 30 min, the reaction was quenched with Tris (pH 6.8), to a final concentration of 50 mm. Products were analyzed by SDS-PAGE and visualized by Coomassie Brilliant Blue R250 staining. Trials were also performed with glutaraldehyde (Sigma) and disuccinimidyl glutarate (DSG), obtained from Pierce. The latter is a homobifunctional reagent of the N-hydroxysuccinimide ester family that also targets primary amino groups (32Ji T.H. Methods Enzymol. 1983; 91: 580-609Crossref PubMed Scopus (129) Google Scholar). With these reagents, the protocol above was used with the following exceptions: glutaraldehyde was added as an aqueous solution to a final concentration of 0.1%; and DSG to 500 μm, from a freshly prepared 10 mm stock in Me2SO (Me2SO contributed no more than 5% of the total reaction volume of 20 μl). Gel filtration chromatography is used in our laboratory in the purification of UvrB. This is performed at 4 °C using a 4.9 × 110-cm column of Sephacryl S-300, equilibrated with 50 mm potassium MOPS, pH 7.5, 500 mm KCl, 2 mm β-mercaptoethanol, 15% glycerol. Molecular weight standards were run through this column, in duplicate, as a routine performance evaluation. Comparison of the single elution peak of UvrB from two purification runs with a calibration plot of the partition coefficients determined for the molecular weight standards yielded a predicted Stokes radius of 4.55 nm, and an apparent molecular weight estimate of 150,000. This suggested that, under the conditions described, UvrB occurred predominantly as a dimer (if roughly globular, as would be expected for the standards). More rigorous gel filtration studies were performed with a 1.0 × 30-cm Superose 12 fast protein liquid chromatography column at room temperature in a buffer of 50 mm HEPES, pH 7.6, 300 mm KCl, 10 mm MgCl2, 5% glycerol, 2 mm DTT. The performance of this column can be judged from the calibration curve shown in Fig.1 a, in which the log of the Stokes radius for standards ranging in molecular weight from 12,400 to 669,000 is plotted versus the observed partition coefficientK av (error estimates for the mean partition coefficients for standards and protein samples expressed as the coefficient of variation ranged from 0.21 to 1.94%; the regression correlation coefficient, R2, for the calibration curve was 0.979). Loaded onto this column at initial concentrations ranging from 1 to 35 μm (1 μm being the lower limit for a significant signal with our apparatus), UvrB eluted consistently as a single peak with minor tailing but with the peak position showing a strong concentration dependence. The effective radii of hydration (Stokes radius) for UvrB at the differing initial concentrations were calculated from the calibration curve shown and are plotted in Fig.1 b. At the lowest concentration, 1 μm, the partition coefficient coincided with a Stokes radius of 3.7 nm or an apparent molecular weight of 97,000 (estimated from an alternate calibration curve plotting K av versus M r). At an initial concentration of approximately 24 μm, the partition coefficient approached an asymptotic value corresponding to a Stokes radius of 4.67 nm or apparent molecular weight of 170,000. These results are indicative of associative behavior and suggest a monomer-dimer equilibrium (molecular weights predicted from the amino acid composition are 76,091 and 152,182, respectively). The single peak observed at any given concentration further suggests that equilibration is rapid with respect to the limits imposed by the elution experiment. The higher values for molecular weights obtained by gel filtration, compared with those predicted by sequence, could derive from a number of factors. The lower molecular weight predicted represents, of course, a limit of detection imposed by our experimental conditions; a lower plateau was not demonstrated but would presumably be intermediate between that observed and the value estimated for UvrB* (below). Partitioning in gel permeation, furthermore, is a function of molecular shape as well as size (27Potschka M. Anal. Biochem. 1987; 162: 47-64Crossref PubMed Scopus (122) Google Scholar), hence deviation from the globular shape of the commonly used standards could lead to error. Interaction of protein with the gel matrix cannot be excluded, though the salt concentration used (300 mm) should minimize this possibility. Finally, the observed partition coefficient represents a mass averaged function of the species in equilibrium. A minor population of higher order oligomers could skew the partition function toward values suggesting a higher molecular weight. In contrast to UvrB, the elution behavior of UvrB* provides no indication of a potential for oligomeric self-association. Over a range in initial concentration from 1 to 30 μm, the partition coefficient corresponds to a Stokes radius of 3.4 nm, or apparent molecular weight of 77,000. Depending on which cleavage site is recognized by the ompT protease (20Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 33Arikan E. Kulkarni M.S. Thomas D.C. Sancar A. Nucleic Acids Res. 1986; 14: 2637-2650Crossref PubMed Scopus (58) Google Scholar), the molecular weight predicted from sequence for a monomer of UvrB* would be in the range of 68,588 to 71,080. A single elution peak was also observed for UvrB*, except at the highest concentration, where a minor peak coincided with the column void volume. It is likely that this material resulted from a large-scale aggregation of UvrB*. Such aggregates would have a Stokes radius in excess of that of the largest protein standard used, M r 670,000, and would likely include ten subunits as a minimum. The propensity of UvrB* to form large aggregates in concentrated solution had been surmised from its excessive but variable ability to scatter light, as observed (data not shown) both in absorbance and fluorescence emission spectra (Rayleight scatter peaks). A tendency to form such aggregates has been reported both for UvrA and UvrC (6Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Crossref PubMed Scopus (167) Google Scholar, 34Yeung A.T. Mattes W.B. Oh E.Y. Yoakum G.H. Grossman L. Nucleic Acids Res. 1986; 14: 8535-8556Crossref PubMed Scopus (38) Google Scholar). Electrophoresis through a nondenaturing polyacrylamide gel offers a relatively rapid yet sensitive technique for examination of the potential for self-assembly by a protein. Multiple bands can be resolved when UvrB is electrophoresed on such gels, the most useful to date being a 4–30% gradient gel, as shown in Fig. 2. Run at room temperature, five bands can be discerned (and corroborated by densitometric profiles, not shown) when 15 μg of UvrB is applied (lane 2). Three bands remain detectable by Coomassie staining with the protein load reduced to 5 μg (gel not shown). An equal loading (i.e. 15 μg) of this protein on SDS gels gives a single band when visualized by Coomassie staining, with an estimate of 98–99% purity from densitometer scans (cross-linking gels to be shown below). That the state of assembly is temperature-dependent is evident by comparison with the migration profiles seen in Fig. 2, lanes 5 and 6. With electrophoresis performed at 4 °C rather than at room temperature, the number of visible UvrB bands (lane 5) is apparently reduced (three are detectable; that of highest molecular weight marginal by eye but detectable by densitometry), the distribution is altered to more strongly favor the band with greatest mobility (68% of lane density, as compared with 36% at 22 °C), and the mobility of this major band is shifted relative to that of the molecular weight standards (toward an apparently higher molecular weight). It is our suggestion that this most mobile band of UvrB is not populated by monomeric UvrB, but by monomer and dimer in rapid equilibrium and with the equilibrium position altered by temperature. If so, the additional bands may represent oligomers with from three to six subunits. Assignment of the degree of oligomerization to bands is not straightforward. The mobility of a given band in a nondenaturing gel depends on molecular charge as well as size and shape. By use of a gradient gel with up to 30% polyacrylamide, protein standards in a size range appropriate to the study of UvrB oligomerization will, if driven long enough by an applied voltage, reach a pore size small enough to block further migration. If electrophoresis is continued until all proteins, even those with low charge density, reach their terminal postions, then comparison of sample mobility with that of standards has merit (35Margolis J. Wrigley C.W. J. Chromatogr. 1975; 106: 204-209Crossref Scopus (76) Google Scholar). The use of standards, however, to estimate the molecular weight of a sample is subject to uncertainty unless it is known that the relation of both size and shape to molecular weight is invariant between sample and standards (36Felgenhauer K. Hoppe-Seyler's Z. Physiol. Chem. 1974; 355: 1281-1290Crossref PubMed Scopus (56) Google Scholar). Given this caveat, the estimated apparent molecular weights for the UvrB bands marked by arrows in Fig. 2, lane 2 (22 °C) are: 106,800 ± 4,100, 201,200 ± 11,900, 263,700 ± 10,800, 318,600 ± 9,100, and 394,800 ± 6,700. On their own, and not unexpectedly, these estimates do not permit unambiguous assignment of the bands. The estimate for the smallest UvrB species is intermediate between that of a monomer (76,091) and of a dimer (152,182). When run at 4 °C, this band migrates with an apparent molecular weight closer to that expected for the dimer, 145,400 ± 6,600. In gradient gels, the leading edge of the band, where molecules would encounter smaller pore sizes, is usually sharpened. The broadness of the major UvrB bands, at either temperature, and the diffuse appearance of the leading edge, in addition to the temperature dependence support the suggestion that these bands may represent a weight-averaged migration position of monomer and dimer in rapid equilibrium. This interpretation gains support from the single elution peak noted in the gel filtration studies with UvrB. If so, the slower migrating bands could be assigned to tri-, tetra-, penta-, and hexamers; with these assignments the estimates of apparent molecular weight would underestimate the values calculated from the amino acid composition by 12–16%. With proteolytic truncation of the C terminus, the potential for self-association of UvrB is significantly reduced, yet potential oligomer bands can be seen when 15 μg of UvrB* is electrophoresed in native gels (Fig. 2, lanes 3and 6). The major band has an estimated molecular weight of 70,500 ± 2,200 (22 °C run, lane 3) to 73,700 ± 1,500 (4 °C run, lane 6). This is close to that predicted for a monomer from the amino acid composition, approximately 68,500–71,080, depending, as noted above, on the site of cleavage. Cleavage at more than one site may in fact account for the additional bands not marked by arrows flanking the major UvrB* band, although contaminants derived from the exposure to ompT-expressing cells cannot be ruled out. The major band itself, on close examination, appears to be a doublet. Conceivably, this may result from near equal cleavage rates at either lysine 605 or 607, generating products differing in molecular mass by 230 Da, as recently suggested (20Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The mobility of the putative oligomers of UvrB*, denoted also by open arrowheads, were used to estimate molecular weights ranging from 113,000 ± 6,500 (4 °C gel) to 138,100 ± 10,200 (22 °C), a potential dimer, and from 172,400 ± 11,000 (22 °C) to 216,000 ± 9,200 (4 °C), a potential trimer. The potential for self-association of UvrB and UvrB* was further examined with a nonhydrodynamic approach, chemical cross-linking. The purity of the UvrB protein used in experiments was estimated to be 98–99%, as judged by densitometer scans (Fig. 3, lane 2 and from replicate experiments). Covalently linked oligomers of UvrB, resolved by SDS-PAGE, were readily obtained with the lysine-reactive bifunctional reagents glutaraldehyde, DSG, and DMS. Results with DMS are shown in Fig. 3. With exposure of 5 μm UvrB for 30 min to 5 mg/ml of DMS, cross-linked products included apparent dimers (migration position marked by the solid arrow) and higher order oligomers. Though UvrB is a cryptic DNA-dependent ATPase, we recently demonstrated that it binds ATP, with an apparentK D ∼ 1 mm (23Hildebrand E.L. Grossman L. J. Biol. Chem. 1998; 273: 7818-7827Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Cross-linking efficiency was not, however, significantly altered by the addition of Mg-ATP to 8 mm. Variation in the potassium chloride concentration, from 25 to 300 mm, also was without effect on the yield of dimers or total cross-linked species, suggesting that the self-association of UvrB is largely hydrophobic in character. We do note for future study, though, that the distribution of the higher order oligomers may be dependent on salt concentration. As the KCl concentration is increased" @default.
• W2007411488 created "2016-06-24" @default.
• W2007411488 creator A5026416444 @default.
• W2007411488 creator A5048476730 @default.
• W2007411488 date "1999-09-01" @default.
• W2007411488 modified "2023-09-30" @default.
• W2007411488 title "Oligomerization of the UvrB Nucleotide Excision Repair Protein of Escherichia coli" @default.
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