Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B
2007; Elsevier BV; Volume: 282; Issue: 14 Linguagem: Inglês
10.1074/jbc.m609632200
ISSN1083-351X
AutoresMichael G. Kemp, Brian Bae, John Paul Yu, Maloy Ghosh, Michael Leffak, Satish K. Nair,
Tópico(s)RNA modifications and cancer
ResumoLocal zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0Å crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both d-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA. Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0Å crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both d-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA. During each division of somatic cells DNA replication is regulated so that the genome is copied in its entirety only once (1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1394) Google Scholar, 2Schwob E. Curr. Opin. Microbiol. 2004; 7: 680-690Crossref PubMed Scopus (25) Google Scholar). The classical replicon hypothesis states that the initiation of DNA replication at replication origins is controlled by initiator protein binding at regulatory sites called replicators and has served as a useful description for prokaryotic replication (3Jacob F. Brenner S. Cuzin F. Cold Spring Harbor Symp. Quant. Biol. 1963; 28: 329-348Crossref Google Scholar). In contrast, a concise description of eukaryotic replication is complicated by the fact that eukaryotic chromosomes contain multiple origins of replication and the replicator sequences controlling them do not share easily recognizable conserved sequences (4Gilbert D.M. Science. 2001; 294: 96-100Crossref PubMed Scopus (228) Google Scholar, 5Mechali M. Nat. Rev. Genet. 2001; 2: 640-645Crossref PubMed Scopus (96) Google Scholar). 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Michelotti E.F. Pullner A. Duncan R.C. Eick D. Levens D. Mol. Cell. Biol. 1996; 16: 2656-2669Crossref PubMed Scopus (196) Google Scholar), which has been shown to be sensitive to single strand-directed reagents in vitro and in vivo (19Berberich S. Trivedi A. Daniel D.C. Johnson E.M. Leffak M. J. Mol. Biol. 1995; 245: 92-109Crossref PubMed Scopus (44) Google Scholar, 27Michelotti G.A. Michelotti E.F. Pullner A. Duncan R.C. Eick D. Levens D. Mol. Cell. Biol. 1996; 16: 2656-2669Crossref PubMed Scopus (196) Google Scholar). Deletion of the DUE/ARS region eliminates c-myc origin activity (13Liu G. Malott M. Leffak M. Mol. Cell. Biol. 2003; 23: 1832-1842Crossref PubMed Scopus (71) Google Scholar), and a heterologous DUE restores origin activity, 4G. Liu and M. Leffak, unpublished information. implying that a DUE is essential for chromosomal replication origin activity. In addition to a region of easily unwound DNA, replication origins frequently contain binding sites for auxiliary factors that recruit constituents of the replication complex. Yeast one-hybrid studies utilizing the DUE from the c-myc origin as bait identified a 24-kDa polypeptide capable of specifically engaging the DUE in vivo (28Ghosh M. Kemp M. Liu G. Ritzi M. Schepers A. Leffak M. Mol. Cell. Biol. 2006; 26: 5270-5283Crossref PubMed Scopus (30) Google Scholar) and in vitro in the presence of other nuclear proteins (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This protein, termed DUE-B, is conserved in bacteria, yeasts, and eukaryotes with homologs identified in rodents, amphibians, and fish. Recombinant DUE-B purified from baculovirus-infected insect cells is a homodimer that co-purifies with ATPase activity (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In HeLa cells, short interfering RNA-mediated knock down of DUE-B delays entry into S phase and promotes cell death. Moreover, immunodepletion of DUE-B from Xenopus egg extracts inhibits DNA replication and addition of purified, recombinant DUE-B expressed in HeLa cells restores replication activity to these extracts, emphasizing the interspecies conservation of DUE-B function (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). These observations provide strong evidence that DUE-B is involved in the initiation of DNA replication. To gain further insights into the function of DUE-B in DNA replication, we have solved the three-dimensional crystal structure of the recombinant protein to a resolution of 2.0 Å and, guided by our crystal structure, have carried out further biochemical characterization of DUE-B. Homodimerization of DUE-B is mediated by an extensive set of interactions that result in the formation of a continuous β-sheet structure between the two monomers. The C-terminal residues that are involved in mediating interactions with target DNA are largely disordered in the crystal, in agreement with results from limited proteolysis experiments. The three-dimensional structure of DUE-B reveals notable similarity to the overall domain structures of both d-aminoacyl-tRNA deacylases and the archaea-specific editing domain of threonyl-tRNA synthetase (30Ferri-Fioni M.L. Schmitt E. Soutourina J. Plateau P. Mechulam Y. Blanquet S. J. Biol. Chem. 2001; 276: 47285-47290Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 31Lim K. Tempczyk A. Bonander N. Toedt J. Howard A. Eisenstein E. Herzberg O. J. Biol. Chem. 2003; 278: 13496-13502Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 32Dwivedi S. Kruparani S.P. Sankaranarayanan R. Nat. Struct. Mol. Biol. 2005; 12: 556-557Crossref PubMed Scopus (45) Google Scholar). In vitro analyses confirm that DUE-B possesses both d-aminoacyl-tRNA deacylase and ATPase activities. Purification and Crystallization—Recombinant human DUE-B containing a C-terminal His6 tag (rDUE-B) was purified from baculovirus-infected insect cells (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Forty-eight hours after infection the cells were lysed and DUE-B purified by Ni+2-NTA resin affinity chromatography (Qiagen) according to the manufacturer's instructions. Fractions containing >95% pure rDUE-B (200 mm imidazole eluate) were identified by SDS-PAGE and silver staining. Protein was concentrated and buffer exchanged into 10 mm Tris-Cl, pH 7.6, 100 mm NaCl, 10% glycerol by centrifugal filtration (Millipore). Immediately prior to crystallization, protein samples were exchanged into a buffer composed of 100 mm NaCl and 10 mm HEPES, pH 7.5, by ultrafiltration. Crystals suitable for diffraction studies were obtained by the hanging drop method by mixing 2 μl of protein (6 mg/ml) with 2 μl of a well solution (200 mm KCl, 50 mm sodium cacodylate, pH 6.5, and 10% (w/v) polyethylene glycol 8000) and equilibrating against the latter solution at 12 °C. For cryo-crystallography, crystals were serially transferred into the precipitant solution containing incremental concentrations of glycerol up to a final concentration of 30% and flash-cooled by plunging directly into liquid nitrogen. Native diffraction data were collected at an insertion device synchrotron source using an ADSC CCD detector (Beamline 17-ID, Advanced Photon Source, Argonne National Laboratory, Argonne, IL) at 100 K. Data were indexed and scaled using HKL2000 (33Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar) to a limiting resolution of 2.0 Å. A derivative data set was collected at 100 K from crystals soaked in 1 mm ethylmercuric phosphate using a RAXIS IV++ image plate detector system to a limiting resolution of 3.0 Å. The data collection and processing statistics are summarized in Table 1.TABLE 1Data collection, phasing, and refinement statisticsNativeDerivative (ethylmercuric phosphate)Data collection Space groupP31P31 Cell dimensions a, b, c (Å)77.8, 77.8, 106.477.9, 77.9, 108.3 α, β, γ (°)90.0, 90.0, 120.090.0, 90.0, 120.0 Resolution (Å)50-2.0 (2.05-2.0)aHighest resolution shell is shown in parentheses30-3.0 Rsym (%)4.0 (28.2)8.9 (32.0) I/σI39.9 (6.2)11.5 (4.0) Completeness (%)99.8 (100.0)99.0 (94.7) Redundancy6.4 (5.9)3.8 (3.6)Refinement Resolution (Å)25.0-2.0 No. reflections45,864 Rwork/Rfree20.4/23.7 No. atoms Protein4713 Magnesium ion4 Water360 B-factors Protein44.09 Magnesium ions36.32 Water46.35 Root mean square deviations Bond lengths (Å)1.44 Bond angles (°)0.014a Highest resolution shell is shown in parentheses Open table in a new tab Phasing, Model Building, and Refinement—Initial crystallographic phases were obtained from a mercurial derivative (34Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1937-1940Crossref PubMed Scopus (283) Google Scholar, 35Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Although the resultant electron density was not readily interpretable, solvent flattening and non-crystallographic symmetry averaging (36Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar) between the multiple copies of DUE-B in the crystallographic asymmetric unit allowed for an initial trace of the protein main chain atoms. Multiple rounds of 2- and 4-fold non-crystallographic symmetry averaging (36Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar), followed by cycles of manual rebuilding and crystallographic refinement using REFMAC5 (37Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13868) Google Scholar), allowed assignment of the first 151 amino acids of each of the four monomers in the crystallographic asymmetric unit. Refinement against the 2.0 Å synchrotron data yielded a final model with a working R factor of 20.4% and a free R factor of 23.7%. The final model contains the first 151 amino acid residues for each DUE-B monomer, 379 water molecules, and 4 magnesium ions. The refined coordinates have been deposited in the Protein Data bank with accession number 2OKV. Enzyme Assays—Protease digestions of DUE-B were performed with trypsin, chymotrypsin, or V8 protease at 1:20–1: 400 w:w protease:DUE-B. Aminoacylation of Escherichia coli tRNA was performed as described by Soutourina et al. (38Soutourina J. Blanquet S. Plateau P. J. Biol. Chem. 2000; 275: 11626-11630Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) to minimize contamination by l-aspartic acid. A 500-μl reaction containing 100 mm Tris, pH 7.6, 5 mm MgCl2, 50 mm KCl, 0.5 mm EDTA, 2.5 mm ATP, 1% glycerol, 0.6 mm β-mercaptoethanol, 5 μm d-[3H]aspartic acid (PerkinElmer), 25 μm E. coli tRNA (Sigma), and 1250 units of mixed E. coli tRNA synthetases (Sigma) was incubated for 10 min at 37 °C. After overnight precipitation with ethanol at -20 °C, the supernatant was lyophilized, resuspended in water, and used again in an aminoacylation reaction at 37 °C for 90 min with an additional 12.5 nmol E. coli tRNA and 1250 units of E. coli tRNA synthetase. The reaction was extracted with phenol-chloroform, precipitated with ethanol, and used fresh in deacylation reactions. E. coli tRNA (Sigma) aminoacylated with d-[3H]aspartate was incubated in a 50-μl reaction containing 20 mm Tris-HCl, pH 7.8, 5 mm MgCl2, and 500 fmol of the indicated form of recombinant DUE-B. The reaction was incubated at 37 °C for 10 min before addition of glycogen and precipitation with ethanol at -20 °C overnight. After centrifugation the supernatant was analyzed by liquid scintillation counting. To assay for ATPase activity, reactions containing 25 mm Tris, pH 7.6, 1 mm dithiothreitol, 0.2 mg/ml bovine serum albumin, 5 mm MgCl2, 100 μm unlabeled ATP, 100 nm [γ-32P]ATP (PerkinElmer), and the indicated amount of recombinant DUE-B were incubated at 37 °C for 60 min. Reactions were stopped by addition of EDTA to 50 mm and frozen by dropping into liquid N2. An aliquot of each reaction was spotted on a polyethyleneimine (PEI)-cellulose TLC plate (Selecto Scientific) that was developed in 0.8 m acetic acid/0.8 m LiCl and analyzed using a PhosphorImager. To assay for DUE-B dimer stability, rDUE-B was mixed with Xenopus egg extract for 40 min at room temperature. The mixture was subsequently incubated with Ni+2-NTA-agarose beads for 4 h at 4 °C. Electrophoretic Mobility Shift Assays—Recombinant forms of DUE-B containing or lacking the C terminus were used in electrophoretic mobility shift assays to determine DNA binding. The DNA probe was generated using PCR in the presence of [α-32P]dCTP with primers amplifying a 123-bp region of the c-myc replicator. Preparation of the protease-resistant DUE-B core by trypsin digestion (1:100 w:w trypsin:DUE-B) was as described, except the reaction volume was 10 μl and contained 25 pmol DUE-B. After 1 h at 37 °Ca 0.5-μl aliquot was removed for SDS-PAGE analysis, and the remaining reaction mixture was brought up to 15 μl with TBE (Tris borate-EDTA) (0.5×), NaCl (50 mm), loading dye (1× final concentration; Promega), and 25 fmol 32P-labeled PCR product. Samples were then loaded on a 4% native polyacrylamide (0.5× TBE) gel that had been pre-run for 30 min at room temperature. The gel was run at 100 V for 50 min at room temperature, dried, and analyzed by autoradiography. Details of Overall Topology—We have solved the crystal structure of human DUE-B to 2.0 Å resolution using non-crystallographic symmetry averaging of phases determined from a mercurial derivative (Table 1). Although mass spectrometric analysis of dissolved crystals of DUE-B documents that crystals contain the full-length polypeptide encompassing all 209 residues, interpretable electron density is only visible for the first 151 amino acids. Predictive analysis and limited proteolysis studies (see below) are consistent with the view that the final 58 amino acid residues are unstructured in the absence of additional ligands. The N-terminal 151 residues of DUE-B fold into a compact domain of the α/β class of proteins (Fig. 1a). Residues Lys-2 through Val-15 form a long, extended β strand (β1) that is sharply bent at residue Thr-9. Two β strands, encompassing residues Glu-18 through Ile-23 (β2) and residues Gly-26 through Gly-32 (β3), form the core of the β-sheet structure. Residues Gln-39 through Asn-51 form helix α1, followed by the next strand (β4) of the sheet structure created by Glu-73 through Ser-78. This is followed by a second long helix (α2) encompassing residues Thr-99 through Thr-116. Residues Ile-122 through Asp-124 form a short β strand (β5). The long, final β strand (β6), created by residues Met-131 through Glu-146, with a kink at residue Gly-139, completes the structure of this domain of DUE-B (see Fig. 2 for a secondary structure annotation of the DUE-B monomer).FIGURE 2Structure-based sequence alignment. Multiple sequence alignment of the primary sequence of human DUE-B (with the corresponding secondary structure elements annotated above the sequence) with the sequences of eukaryotic homologs. Residues implicated to be involved in catalysis are shown as cyan diamonds, and the juncture sensitive to protease or chemical cleavage is shown with orange stars.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Solution studies show that DUE-B is a homodimer (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), and this dimeric interaction is observed in the crystal structure. Within the asymmetric unit, there are four copies of the DUE-B monomer. Pairs of monomers form tight interacting dimer, yielding two copies of the DUE-B homodimer in the asymmetric unit. Each homodimer buries a total of 2295 Å2 of surface area. The dimer interface is created mainly by intermolecular interactions between strandβ6 residues Met-131 through Gly-139 from each monomer to create an antiparallel β-sheet structure that extends across the homodimer interface (Fig. 1b). The primary sequence of human DUE-B shows strong evolutionary conservation of sequences found in higher and lower eukaryotes and in bacteria (Fig. 2). The topological arrangement of the DUE-B monomer bears many of the structural elements of polypeptides that contain nucleotide binding motifs (Fig. 3). Structure-based comparison of DUE-B with the ATP-binding protein MJ0577 (1MJH) (39Zarembinski T.I. Hung L.W. Mueller-Dieckmann H.J. Kim K.K. Yokota H. Kim R. Kim S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15189-15193Crossref PubMed Scopus (275) Google Scholar) yields a root mean square deviation of 3.7 Å over 79 residues (Fig. 3b). A structural comparison with liver alcohol dehydrogenase demonstrates that the DUE-B N-terminal domain structure is similar to half of the canonical Rossman fold (Fig. 3c). A superposition of 62 α carbon atoms common to DUE-B and alcohol dehydrogenase (1A4U) (40Benach J. Atrian S. Gonzalez-Duarte R. Ladenstein R. J. Mol. Biol. 1998; 282: 383-399Crossref PubMed Scopus (95) Google Scholar) yields a root mean square value of 3.7 Å. Given the observation that recombinant DUE-B possesses the ability to hydrolyze ATP (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), it is quite likely that the structural similarity between the N-terminal domain of DUE-B and these proteins extends to its function as a nucleotide binding motif, as illustrated below. Stability of the DUE-B Dimer— DUE-B isolated from Xenopus egg extract (xDUE-B), HeLa nuclei, or human recombinant DUE-B expressed in insect cells (rDUE-B) is in the form of a homodimer (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). When rDUE-B was mixed with Xenopus egg extract and reisolated by binding to Ni+2-NTA-agarose, rDUE-B did not co-isolate with xDUE-B (Fig. 4a), implying that the rDUE-B and xDUE-B dimers do not disproportionate in vitro. Similarly, rDUE-B did not dimerize with HeLa DUE-B when mixed in vitro with HeLa nuclear extracts (not shown). In contrast, when His6-tagged human DUE-B was expressed in HeLa cells and purified by Ni+2-NTA-agarose, the ectopic and endogenous forms of DUE-B co-isolated in extracts from cells arrested in G1, S, or M phase of the cell cycle (Fig. 4b). These results suggest that the DUE-B heterodimers and, by extension, DUE-B homodimers formed in vivo are stable over the cell cycle. The crystal structure of DUE-B suggests that the C-terminal portion of the protein is not involved in dimerization. To test this directly, a C-terminal truncation mutant missing amino acids 148–209 was expressed in HeLa cells. As shown in Fig. 4c, the truncated form of DUE-B efficiently dimerized with the endogenous wild type protein. Taken together, these results show that DUE-B dimers are stable in vivo and in vitro and that the dynamic C-terminal region is not essential for dimerization. Protease Sensitivity of the DUE-B C Terminus and DNA Binding—The C-terminal portion of DUE-B (amino acids 152–209) is not visible in the crystal structure and is predicted to be dynamically disordered (41Iakoucheva L.M. Kimzey A.L. Masselon C.D. Bruce J.E. Garner E.C. Brown C.J. Dunker A.K. Smith R.D. Ackerman E.J. Protein Sci. 2001; 10: 560-571Crossref PubMed Scopus (101) Google Scholar, 42Romero P. Obradovic Z. Dunker A.K. Appl. Bioinformatics. 2004; 3: 105-113Crossref PubMed Scopus (126) Google Scholar, 43Li X. Romero P. Rani M. Dunker A.K. Obradovic Z. Genome Inform. Ser. Workshop Genome Inform. 1999; 10: 30-40PubMed Google Scholar). Trypsin or chymotrypsin digestion of His6-tagged human DUE-B (rDUE-B) purified from baculovirus-infected Sf9 insect cells produced a resistant core of ∼17 kDa, close to the predicted size of the monomeric N-terminal domain visible in the DUE-B crystal (Fig. 5a). Protease digestion removed the C-terminal His6 tag during production of the resistant core (Fig. 5b), and formic acid cleavage of full-length DUE-B between Asp-156 and Pro-157, or formic acid treatment of trypsin-digested DUE-B, resulted in a product of closely similar size to that of the protease-resistant core (Fig. 5c), demonstrating that it is the C terminus that is removed by proteolysis. Disordered regions of DNA-binding proteins often become ordered upon DNA binding (42Romero P. Obradovic Z. Dunker A.K. Appl. Bioinformatics. 2004; 3: 105-113Crossref PubMed Scopus (126) Google Scholar). Incubation of rDUE-B with a 54- or 123-bp DNA fragment containing the DUE (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) resulted in the resistance of the full-length protein to protease digestion (Fig. 6a). Comparative time course digestions of DUE-B in the absence and presence of DNA (Fig. 6b) revealed a significant (4- to 5-fold) delay in susceptibility to digestion in the presence of the polynucleotide ligand. Consistent with the lack of sequence specificity of purified DUE-B binding (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), the DUE was not essential for resistance to trypsin digestion, as poly(dI-dC)·poly(dI-dC) fully protected DUE-B and mixed E. coli tRNAs moderately protected DUE-B against trypsin digestion (not shown). By contrast, a 54-nt single-stranded oligonucleotide containing one strand of the DUE or a 21-nt oligonucleotide from the DUE/ARS segment did not afford significant protection against digestion (Fig. 6c). Because DUE-B was isolated by virtue of its binding to the c-myc DUE in vivo and selectively binds a double-stranded DNA fragment containing the DUE in vitro in the presence of HeLa nuclear extract (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), the observation that double-stranded DNA preferentially protects the C terminus of DUE-B against protease suggests that in the presence of in vivo binding partners DUE-B may recognize some feature of incipiently unwound DNA prior to actual strand separation. Purified full-length rDUE-B is able to bind double-stranded DNA without sequence specificity in an electrophoretic mobility shift assay (29Casper J.M. Kemp M.G. Ghosh M. Randall G.M. Vaillant A. Leffak M. J. Biol. Chem. 2005; 280: 13071-13083Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). As shown in Fig. 6d, to test whether the C terminus of DUE-B is necessary for DNA binding, the electrophoretic mobility shift assay was repeated using the protease-resistant DUE-B core, the C-terminal truncation mutant, or the trypsin-treated C-terminal truncation mutant (which is resistant to trypsin digestion, as expected from the results of Fig. 5). Full-length rDUE-B was able to form a stable complex with the double-stranded DNA probe whereas neither the protease-resistant core nor the C-terminal truncation mutant bound to the probe, implying that the C terminus is necessary for binding of purified DUE-B to DNA. Structural Similarities to tRNA-editing Enzymes—Human DUE-B shows strong evolutionary conservation with tRNA-binding proteins in yeasts and metazoans. The overall conformation of the N-terminal 151-amino acid domain of DUE-B bears significant similarity to the nucleotide binding Rossman fold found in aminoacyl-tRNA synthetases (44Park S.G. Ewalt K.L. Kim S. Trends Biochem. Sci. 2005; 30: 569-574Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) and demonstrates significant homol
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