The Yeast STM1 Gene Encodes a Purine Motif Triple Helical DNA-binding Protein
2000; Elsevier BV; Volume: 275; Issue: 8 Linguagem: Inglês
10.1074/jbc.275.8.5573
ISSN1083-351X
AutoresLaura D. Nelson, Marco Musso, Michael W. Van Dyke,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoThe formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. Using conventional and affinity chromatography, we purified from Saccharomyces cerevisiaewhole-cell extract a 35-kDa protein that avidly and specifically bound a purine motif triplex (with a K d of 61 pm) but not a pyrimidine motif triplex or duplex DNA. Peptide microsequencing identified this protein as the product of theSTM1 gene. Confirmation that Stm1p is a purine motif triplex-binding protein was obtained by electrophoretic mobility shift assays using either bacterially expressed, recombinant Stm1p or whole-cell extracts from stm1Δ yeast. Stm1p has previously been identified as G4p2, a G-quartet nucleic acid-binding protein. This suggests that some proteins actually recognize features shared by G4 DNA and purine motif triplexes, e.g. Hoogsteen hydrogen-bonded guanines. Genetically, the STM1 gene has been identified as a multicopy suppressor of mutations in several genes involved in mitosis (e.g. TOM1,MPT5, and POP2). A possible role for multiplex DNA and its binding proteins in mitosis is discussed. The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. Using conventional and affinity chromatography, we purified from Saccharomyces cerevisiaewhole-cell extract a 35-kDa protein that avidly and specifically bound a purine motif triplex (with a K d of 61 pm) but not a pyrimidine motif triplex or duplex DNA. Peptide microsequencing identified this protein as the product of theSTM1 gene. Confirmation that Stm1p is a purine motif triplex-binding protein was obtained by electrophoretic mobility shift assays using either bacterially expressed, recombinant Stm1p or whole-cell extracts from stm1Δ yeast. Stm1p has previously been identified as G4p2, a G-quartet nucleic acid-binding protein. This suggests that some proteins actually recognize features shared by G4 DNA and purine motif triplexes, e.g. Hoogsteen hydrogen-bonded guanines. Genetically, the STM1 gene has been identified as a multicopy suppressor of mutations in several genes involved in mitosis (e.g. TOM1,MPT5, and POP2). A possible role for multiplex DNA and its binding proteins in mitosis is discussed. pyrimidine purine triplex-binding protein electrophoretic mobility shift assay polyacrylamide gel electrophoresis triplex-forming oligodeoxyribonucleotide G-quartet fast protein liquid chromatography photo-cross-linked It has long been recognized that, under the proper conditions, certain DNA sequences preferentially adopt a structure composed of three nucleic acid strands (1.Felsenfeld G. Davies D.R. Rich A. J. Am. Chem. Soc. 1957; 79: 2023-2024Crossref Scopus (797) Google Scholar). Triple helical or triplex DNA is a thermodynamically favored structure characterized by a third pyrimidine-rich (Py triplex)1or purine-rich (Pu triplex) DNA strand located within the major groove of a homopurine/homopyrimidine stretch of duplex DNA (reviewed in Ref.2.Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (650) Google Scholar). Both intermolecular triplexes, where the third stand originates from a separate DNA molecule, and intramolecular triplexes (H-DNA), where the third stand originates from a proximal site on the same DNA molecule as its duplex acceptor, have been described. In intermolecular and intramolecular triplexes, stable interaction of the third strand is achieved through either specific Hoogsteen (Py triplex) or reverse Hoogsteen (Pu triplex) hydrogen bonding to the homopurine strand of the duplex, with the third strand adopting either a parallel (Py triplex) or antiparallel (Pu triplex) orientation relative to the homopurine acceptor. Base triplets in the pyrimidine motif include T*AT and C+*GC, whereas those in the purine motif include G*GC, A*AT, and T*AT. Because cytosine protonation requires acidic pH (3.Singleton S.F. Dervan P.B. Biochemistry. 1992; 31: 10995-11003Crossref PubMed Scopus (164) Google Scholar) and the G*GC base triplet is the most stable in the purine motif (4.Beal P.A. Dervan P.B. Nucleic Acids Res. 1992; 20: 2773-2776Crossref PubMed Scopus (107) Google Scholar), T-rich Py motif or G-rich Pu motif triplexes would be expected to predominate under physiological conditions. Do triplexes occur in vivo? Although direct proof is lacking, long oligopurine tracts with triplex-forming potential are quite common in eukaryotic genomes, ranging from yeast to human (5.Behe M. Nucleic Acids Res. 1995; 23: 689-695Crossref PubMed Scopus (107) Google Scholar). These tracts are distributed nonrandomly and are typically located near gene promoters, recombination hot spots, and matrix attachment regions (6.Wells R.D. Collier D.A. Hanvey J.C. Shimizu M. Wohlrab F. FASEB J. 1988; 2: 2939-2949Crossref PubMed Scopus (493) Google Scholar, 7.Boulikas T. J. Cell. Biochem. 1996; 60: 297-316Crossref PubMed Scopus (55) Google Scholar). Additionally, multiple lines of evidence have implicated intramolecular triplexes in several cellular processes, including transcription, replication, and recombination (8.Kohwi Y. Kohwi-Sigamatsu T. Genes Dev. 1991; 5: 2547-2554Crossref PubMed Scopus (85) Google Scholar, 9.Chen S. Supakar P.C. Vellanoweth R.L. Song C.S. Chatterjee B. Roy A.K. Mol. 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Taken together, these data support the existence of triplex DNA at some point during the life cycle of a eukaryotic cell and suggest an important role for these structures in DNA-dependent biological processes. If triplexes form in vivo, whether as required intermediates or as undesired side products of a necessary process, then cellular proteins might also exist that specifically recognize this particular DNA form. To date, four examples of triplex-binding proteins (3BPs) have been described in the literature. These include two reports of similar 55-kDa human proteins that exhibit binding specificity for Py triplex DNAs (16.Kiyama R. Camerini-Otero R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10450-10454Crossref PubMed Scopus (81) Google Scholar, 17.Guieysse A.-L. Praseuth D. Hélène C. J. Mol. Biol. 1997; 267: 289-298Crossref PubMed Scopus (52) Google Scholar), our findings of several human proteins that specifically recognize a Pu triplex (18.Musso M. Nelson L.D. Van Dyke M.W. Biochemistry. 1998; 37: 3086-3095Crossref PubMed Scopus (47) Google Scholar), and evidence that theDrosophila GAGA factor can bind to Py triplexes (but not Pu triplexes) containing a (GA·TC)22 sequence (19.Jiménez-Garcı́a E. Vaquero A. Espinás M.L. Soliva R. Orozco M. Bernués J. Azorı́n F. J. Biol. Chem. 1998; 273: 24640-24648Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Using electrophoretic mobility shift assays (EMSA), we have also found evidence for Pu motif 3BPs in extracts from organisms ranging from bacteria to human. 2M. Musso and L. D. Nelson, unpublished observations. These data suggest that 3BPs are present in all eukaryotes and that they play important cellular roles. To better understand the biological roles of 3BPs, we sought to identify their corresponding genes. We chose the yeastSaccharomyces cerevisiae as our model system, given its completely sequenced genome and the wealth of biochemical and genetic information presently available for this organism (20.Goffeau A. Barrell B.G. Bussey H. Davis R.W. Dujon B. Feldmann H. Galibert F. Hoheisel J.D. Jacq C. Johnston M. Louis E.J. Mewes H.W. Murakami Y. Philippsen P. Tettelin H. Oliver S.G. Science. 1996; 274: 563-567Crossref Scopus (3247) Google Scholar, 21.Hodges P.E. McKee A.H. Davis B.P. Payne W.E. Garrels J.I. Nucleic Acids Res. 1999; 27: 69-73Crossref PubMed Scopus (193) Google Scholar). Here we describe the purification and characterization of the major S. cerevisiae 3BP, y3BP1, and its identification as the product of the STM1 gene. Sequences and structures of duplex, triplex, and quadruplex DNA probes and competitor DNAs used in this study are shown in Fig. 1. Psoralenated oligonucleotides, indicated by a "P" prefix in their name or by a "P∼" appended to their sequence, contained a 4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen-hexyl (Glen Research) moiety attached to their 5′-terminus. All oligonucleotides were purified by n-butanol precipitation (22.Sawadogo M. Van Dyke M.W. Nucleic Acids Res. 1991; 19: 674Crossref PubMed Scopus (110) Google Scholar). Those used in constructing duplex and triplex probes were further purified by denaturing PAGE. Duplex probes and competitor DNAs were made by annealing equimolar (0.1 mm) concentrations of complementary oligonucleotides at room temperature. In the case of labeled probes, annealed duplexes were 3′-end-filled using the Klenow fragment of DNA polymerase and deoxyribonucleotides, including [α-32P]dATP. Duplex DNAs used in this study included the G/C-rich Pu duplex and the A/T-rich Py duplex (Fig. 1). Pu triplexes used in this study contained either the noncovalently attached triplex-forming oligonucleotide (TFO) ODN 1 or the covalently attached psoralenated TFO PODN 1. To form either Pu motif triplex, Pu duplex and a 10-fold molar excess of TFO were incubated for 60 min at 30 °C in a reaction mixture containing 40 mm Tris-HCl (pH 8.0), 100 mmMgCl2, and 0.01% Nonidet P-40 (23.Musso M. Van Dyke M.W. Nucleic Acids Res. 1995; 23: 2320-2327Crossref PubMed Scopus (38) Google Scholar). For pyrimidine motif triplex formation, the Py duplex, TFO PODN 3, and a buffer composed of 25 mm Tris-HCl (pH 6.0), 20 mmMgCl2, and 70 mm NaCl were used instead. Psoralenated TFOs were covalently attached to both strands of the duplex DNA following triplex formation by irradiation at 365 nm for 10 min at 0 °C with a 6-watt hand-held UV lamp. Under these conditions, greater than 90% of the probe is converted to photo-cross-linked triplex (24.Musso M. Wang J.C. Van Dyke M.W. Nucleic Acids Res. 1996; 24: 4924-4932Crossref PubMed Scopus (32) Google Scholar). G-quartet (G4)-containing competitors used in this study included the tetrameric parallel structure GL-tetramer, the intermolecular quadruplex ODN 1 dimer, and the intramolecular quadruplex ODN 1 monomer. Each G4 DNA was formed following published procedures (25.Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Crossref PubMed Scopus (130) Google Scholar, 26.Cheng A.-J. Van Dyke M.W. Gene (Amst.). 1997; 197: 253-260Crossref PubMed Scopus (38) Google Scholar, 27.Cheng A.-J. Wang J.C. Van Dyke M.W. Antisense Nucleic Acid Drug Dev. 1998; 8: 215-225Crossref PubMed Scopus (34) Google Scholar). All DNA probes and competitor DNAs were analyzed by PAGE prior to use. Protein mixtures containing 3BPs were incubated for 30 min at 24 °C in a 10-μl volume containing buffer A (25 mm HEPES-Na+, pH 7.9, 50 mm KCl, 10% glycerol, 0.5 mm dithiothreitol), 1 mmMgCl2, and 0.1 nm probe DNA, together with 2 μg of poly(dI-dC) carrier DNA or additional competitor nucleic acids as indicated. Resulting protein-probe complexes were resolved by nondenaturing PAGE at 7 V/cm for 90 min through a 5% acrylamide, 0.13% bisacrylamide gel containing 22 mm Tris borate, 0.5 mm EDTA. Probe-containing species were visualized by autoradiography and quantitated by a Storm 840 PhosphorImager (Molecular Dynamics). Haploid yeast (S. cerevisiae, strain FY86 α) was cultured in eight 2-liter flasks containing 500 ml of YPD (1% yeast extract, 2% peptone, 1% dextrose), harvested at midlog phase (A 600 = 0.8), and lysed by vortexing with glass beads according to published protocols (28.Dunn B. Wobbe C.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1990: 13.13.1-13.13.9Google Scholar). Proteins were extracted from 18.6 g of lysed cells in 46 ml of buffer B (50 mm NaHPO4, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 5% glycerol). The extract was cleared by sequential centrifugation at 1500 × g for 5 min at 4 °C followed by 12,000 × g for 10 min at 4 °C and stored frozen at −80 °C. The soluble protein concentration was determined to be 16.3 mg/ml in 46 ml of buffer B, using a Bradford dye binding assay (Bio-Rad). All protein manipulation was performed at 4 °C. Triton X-100 (0.05% final) was added to thawed yeast extract (46 ml) and loaded at 12 ml/h onto a 6-ml (1.4 × 3.7 cm) P-11 phosphocellulose column equilibrated in buffer C (20 mm Tris-Cl, pH 7.3, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 10 mmβ-mercaptoethanol, and 0.05% Triton X-100) and 100 mmKCl. Fifty milliliters of column break-through was collected, and the column bed was washed with 50 ml of buffer C plus 100 mmKCl before the proteins were eluted on a gradient of increasing KCl concentration (100 mm--1 m) in buffer C. Thirty-five 1.9-ml fractions were collected, and their conductivities and protein concentrations were determined. Each was analyzed by EMSA (using a covalent Pu triplex probe) to determine the 3BP activities present. Fractions in the range 450–550 mm KCl contained partially purified y3BP1 and were stored frozen at −80 °C. A 0.5-ml photo-cross-linked Pu triplex-DNA affinity column was prepared with CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer's instructions. 500 nmol of Pu duplex DNA (Fig.1 A) possessing a 3′-terminal hexylamine moiety on the G-rich strand was incubated with 1890 nmol of PODN 1 in 10 ml of buffer D (10 mm NaHCO3, pH 8.3, 12 mmMgCl2, and 0.05% Triton X-100) for 4.5 h at 30 °C in the dark to effect triplex formation. The psoralenated third strand was covalently cross-linked to the Pu duplex following irradiation at 365 nm for 40 min at 0 °C using a 6-watt hand-held UV lamp. At least 65% of the triplexes were determined to be photo-cross-linked (data not shown). A 500-nmol sample of covalent Pu triplex in 50 mm NaHCO3 was mixed with 0.33 g (0.5 ml) of activated Sepharose and incubated for 3 h at 24 °C in the dark to allow coupling to occur. The slurry was collected and washed with 10 ml of buffer D, and excess reactive sites blocked with 1 ml of 1 m ethanolamine and 12 mm MgCl2. The ligated column material was gravity packed into a 0.5 × 3.0-cm column and washed with 5 ml of buffer C plus 250 mmKCl and 2 mm MgCl2. A quarter of the partially purified y3BP1 from the phosphocellulose column (1.25 ml) was thawed, pooled, diluted with buffer C to 225 mm KCl, and loaded at 5 ml/h onto the X-Pu triplex-Sepharose column. Breakthrough from this initial load was reapplied to the X-Pu triplex-Sepharose column. Afterward the column was washed with 2 ml of buffer C plus 250 mm KCl and 2 mm MgCl2. Bound proteins were eluted in three sequential steps of buffer C plus 2 mm MgCl2and either 500, 1000, or 1940 mm KCl (1 ml each). Twelve 0.36-ml fractions were collected and assayed by EMSA. Most y3BP1 activity was present in the 1000 mm KCl step. This chromatography was repeated an additional three times with the remaining y3BP1-containing phosphocellulose fractions. All fractions were stored frozen at −80 °C. Y3BP1-containing fractions from all four runs of the Pu triplex-Sepharose column (4.3 ml total) were thawed, pooled, dialyzed (Spectra/Por 4; molecular weight cutoff, 12 kDa) against 100 ml of buffer C plus 90 mm KCl, and filtered through a 0.22-μm Millex-GS filter (Millipore) before loading at a flow rate of 1 ml/min onto tandem 1-ml Mono Q and Mono S FPLC columns (Amersham Pharmacia Biotech) equilibrated in buffer E (25 mmHEPES-Na+, pH 7.9, 0.5 mm EDTA, 0.5 mm dithiothreitol) plus 100 mm KCl. After being loaded and washed with 5 ml of buffer E plus 100 mm KCl, the tandem columns were separated, and the proteins on the downstream Mono S column were eluted with a 10-ml linear gradient of increasing KCl concentration (100–1000 mm) in buffer E. Eleven 0.7-ml fractions were collected and assayed by EMSA and SDS-PAGE (10% polyacrylamide). Nearly pure y3BP1 was obtained in fraction 8 (350 mm KCl). This was stored frozen at −80 °C. Purified y3BP1 (8 μg) obtained from the Mono S column (fraction 8) was fractionated on a 10% SDS-PAGE gel and visualized by brief Coomassie Blue staining, and a 100-mm3 piece of acrylamide containing the major protein band was excised from the gel. This was sent to the Harvard Microchemistry Facility, which determined the sequences of two tryptic peptides, VNQGWGDDK and DVSNLPSLA, by tandem mass spectrometry on a Finnigan LCQ Quadrupole Ion Trap Mass Spectrometer. These peptide sequences were used in a search of the Yeast Proteome Data base (21.Hodges P.E. McKee A.H. Davis B.P. Payne W.E. Garrels J.I. Nucleic Acids Res. 1999; 27: 69-73Crossref PubMed Scopus (193) Google Scholar). Both peptides mapped with complete identity to sequences within the protein encoded by the S. cerevisiae STM1 gene. A bacterially expressed Stm1p fusion with an N-terminal (His)6 sequence was produced using the pV2a expression vector (29.Van Dyke M.W. Sirito M. Sawadogo M. Gene (Amst.). 1992; 111: 99-104Crossref PubMed Scopus (132) Google Scholar). Plasmid pTU151, which contains the full-length STM1 gene, was obtained from Y. Kikuchi (30.Utsugi T. Toh-e A. Kikuchi Y. Biochim. Biophys. Acta. 1995; 1263: 285-288Crossref PubMed Scopus (20) Google Scholar). This gene was amplified by PCR using Vent polymerase (New England Biolabs) and the amplimers 5′-CGG GAT CCA TTT GAT TTG TTA GGT AAC GAC G-3′ and 5′-CGG AAT TCA GGC TTA AGC CAA AGA TGG CAA G-3′. A 400-ng aliquot of agarose gel-purified PCR product was digested with restriction endonucleases EcoRI and BamHI, ligated into the like-digested plasmid pV2a, and subcloned intoEscherichia coli strain XL1-blue (Stratagene). Correct clones were identified by color selection using the β-galactosidase substrate X-gal (Fisher Biotech). Large scale preparation and immobilized metal affinity chromatography of recombinant (His)6-Stm1p was performed as described previously (29.Van Dyke M.W. Sirito M. Sawadogo M. Gene (Amst.). 1992; 111: 99-104Crossref PubMed Scopus (132) Google Scholar). Recombinant Stm1p was further purified by Mono S FPLC chromatography as was described above for the native y3BP1 protein. Nearly pure (His)6-Stm1p was obtained in fraction 12 (350 mm KCl). To determine whether yeast have proteins that specifically recognize Pu triplex DNA, an EMSA of yeast whole-cell extract using a covalently bound Pu triplex probe (Fig.1 B) was performed. This triplex is based on the well characterized 19-mer triplex-forming oligonucleotide ODN 1 (5′-TGGGTGGGGTGGGGTGGGT-3′), which demonstrates strong, sequence-specific binding with an antiparallel orientation to a G-rich, homopurine duplex DNA target (4.Beal P.A. Dervan P.B. Nucleic Acids Res. 1992; 20: 2773-2776Crossref PubMed Scopus (107) Google Scholar). Use of 5′-psoralenated TFO (PODN 1) and a duplex target containing a proximal 5′-TA-3′ sequence allows the formation of a photo-cross-linked triplex (X-Pu triplex) that remains intact even under conditions that normally promote triplex dissociation (23.Musso M. Van Dyke M.W. Nucleic Acids Res. 1995; 23: 2320-2327Crossref PubMed Scopus (38) Google Scholar, 31.Cheng A.-J. Van Dyke M.W. Nucleic Acids Res. 1993; 21: 5630-5635Crossref PubMed Scopus (101) Google Scholar). Incubation of 1 nm radiolabeled X-Pu triplex probe, 2 μg of poly(dI-dC) carrier DNA, and additional competitor nucleic acids as indicated with 4.5-μg proteins from a yeast whole-cell extract for 20 min at room temperature allowed formation of protein-triplex complexes that could be resolved by nondenaturing PAGE and visualized by autoradiography (Fig.2). Under these conditions, nearly complete shifting of the triplex probe into two slower mobility species (C1 and C2) could be observed (compare lanes 1 and2). The major species, C1, had a relative mobility (R F) compared with the free probe of 0.43 and comprised 87% of the total triplex probe, whereas the minor species, C2, had a R F of 0.53 and comprised 11% of the total triplex probe. The specificity of these protein-triplex interactions was demonstrated by competition binding with other nucleic acids. As shown here, the C1 species was reduced to 64% and less than 3% of its normal amount when 100- and 200-fold molar excess unlabeled, noncovalent Pu triplex DNA was present in the binding reaction, respectively (lanes 4 and 5). Formation of complex C2 was inhibited to a similar but lesser extent under these conditions. Note that the competitor triplex did not contain a psoralen photo-cross-link, indicating that this competition was most likely not the result of psoralen photoadduct recognition. Competition with equivalent molar excesses of Pu duplex probe had no effect on the quantities of C2 and C1 species observed (lanes 7 and8). Likewise, competition with 1000-fold molar excesses of the individual oligonucleotides that comprise the Pu triplex,i.e. the G/A- or C/T-rich strands of the Pu duplex or the TFOs ODN 1 or PODN1, had no appreciable effect on C2 or C1 complex formation. Taken together, these data demonstrated that yeast have proteins that specifically recognize a Pu motif triplex. To further characterize proteins that bind Pu triplexes, we purified the protein(s) responsible for the major protein-triplex EMSA complex, C1. A combination of conventional, affinity, and high performance liquid chromatography was employed. Table I outlines this purification, whereas SDS-PAGE and EMSA analyses of the relevant protein fractions are shown in Fig. 3,A and B, respectively. Initially, whole-cell yeast extract was loaded onto a phosphocellulose cation exchanger, and proteins eluted through a gradient of increasing KCl concentration. Note that no glycerol was present in the elution buffers, to minimize viscosity of the eluent and ensure maximal resolution on this column. The proteins responsible for C1 complex formation eluted in the range of 450–550 mm KCl, as determined by an EMSA of the fractions, and they were effectively separated from those responsible for C2 (see Fig. 3 B; compare lanes 3 and5). Partially purified y3BPs were subjected to affinity chromatography using an X-Pu triplex covalently attached to a Sepharose 4B matrix. The y3BPs present in C1 eluted in the 1000 mmKCl step fraction. Note that the overall complexity of this fraction was not greatly changed through this chromatographic step, though the major 50-kDa contaminant, believed to be yeast cytoplasmic elongation factor 1 α (32.Cottrelle P. Thiele D. Price V.L. Memet S. Micouin J.Y. Marck C. Buhler J.-M. Sentenac A. Fromageot P. J. Biol. Chem. 1985; 260: 3090-3096Abstract Full Text PDF PubMed Google Scholar), was effectively removed (Fig. 3 A, comparelanes 4 and 5). This limited purification achieved with the X-Pu triplex-Sepharose column may be due to there being a mixture of Pu duplex and X-Pu triplex sites on this column (estimated 35:65, respectively), the local high molar concentration of these sites present, or the absence of competing sites normally provided by poly(dI-dC) carrier DNA. Final purification was achieved using high performance liquid chromatography. X-Pu triplex affinity-purified C1 3BPs were loaded onto tandem Mono Q and Mono S FPLC columns. These were separated, and the proteins bound to the downstream Mono S column were eluted with a 100–1000 mmlinear gradient of KCl. Triplex binding activity was concentrated in a single 350 mm KCl fraction (Fig. 3 B), which contained a prominent (>80% total protein) 35-kDa polypeptide (Fig.3 A). Using proteins eluted from SDS-PAGE gel slices, we determined that a 35–40-kDa protein was responsible for the C1 shifted species (data not shown). Based on these data, we concluded that the 35-kDa polypeptide present in Mono S fraction 8, referred to as y3BP1, was the sole protein responsible for the major protein-DNA species observed with a cross-linked Pu triplex probe and yeast whole-cell extracts.Table IPurification of y3BP1StepVolumeMassaMass was measured by a Bradford dye binding assay.ActivitybOne unit is equivalent to the amount of binding activity necessary to drive 50% of a labeled X-Pu triplex probe into complex C1 under our standard EMSA reaction conditions.SAYieldFactormlmgunits × 10 6units/mg × 10 3%Yeast extract46.07516.48.51001Phosphocellulose5.714.32.11473317Triplex-Sepharose3.61.71.16471776Mono Q/Mono S0.70.0970.64660010780a Mass was measured by a Bradford dye binding assay.b One unit is equivalent to the amount of binding activity necessary to drive 50% of a labeled X-Pu triplex probe into complex C1 under our standard EMSA reaction conditions. Open table in a new tab Whereas our previous experiments suggested that y3BP1 recognized an intact Pu triplex DNA species, this need not be the case with our covalent X-Pu triplex probe. It is possible that the psoralen photoadduct itself, or a change it induces in the duplex DNA structure, is actually being recognized, as might be expected for a protein involved in DNA repair (33.Lavasani S. Henriksson G. Brant M. Henriksson A. Radulic M. Manthorpe R. Bredberg A. J. Autoimmun. 1998; 11: 363-369Crossref PubMed Scopus (9) Google Scholar). Alternatively, because noncovalent Pu triplexes are inherently unstable under our standard gel electrophoresis conditions (absence of Mg2+ in the gel buffer) (18.Musso M. Nelson L.D. Van Dyke M.W. Biochemistry. 1998; 37: 3086-3095Crossref PubMed Scopus (47) Google Scholar), our probe might be expected to adopt a structure composed of a single-stranded DNA attached at its 5′-end to a duplex DNA, which is somewhat reminiscent of structures found in DNA replication (34.Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (663) Google Scholar). To verify that an intact Pu triplex was actually being recognized by y3BP1, EMSAs were performed with different labeled probes, including the covalent X-Pu triplex, the noncovalent Pu triplex, and the Pu duplex. Note that in this experiment, electrophoresis was performed at 4 °C and at a lower voltage (4 V/cm) to maintain maximal stability of the y3BP1-DNA complex. As shown in Fig. 4, the X-Pu triplex remained stable throughout electrophoresis, as did the y3BP1-triplex complex C1 (lanes 1 and 2). Also, as expected, the noncovalent Pu triplex probe dissociated under these conditions, quantitatively yielding Pu duplex (lane 3). However, a significant fraction of the labeled probe (15%, as compared with 60% with the X-Pu triplex probe) remained intact as part of complex C1, when purified y3BP1 was present in the binding reaction (lane 4). This was likely not the result of binding to the Pu duplex part of the dissociated Pu triplex probe, because no interactions between y3BP1 and the Pu duplex were observed under these conditions (lane 6). Taken together, these and the competition data from Fig. 2 indicate that y3BP1 recognizes an intact purine motif triplex. The y3BP1 protein may bind a Pu triplex specifically, but does it do so with high affinity? To measure its dissociation binding constant, a constant concentration of y3BP1 was incubated in a near physiological buffer (25 mm HEPES-Na+, pH 7.9, 50 mm KCl, 10% glycerol, 0.5 mm dithiothreitol, and 1 mm MgCl2) with a fixed concentration of labeled, covalent X-Pu triplex probe and increasing concentrations of unlabeled, X-Pu triplex DNA. Protein-DNA complexes resulting from these reactions were analyzed by EMSA. Plotting the ratio of bound to free triplex DNA as a function of C1 concentration, we found that y3BP1 exhibited an apparent K d = 61 pm for Pu triplexes (Fig. 5). We also determined that our Mono S fraction 8 contained 1.1 μm active y3BP1, which is comparable to the concentration of a 35-kDa protein estimated from the Coomassie Blue-stained SDS-PAGE gel (3.2 μm). This binding constant is similar to those of many duplex DNA-binding proteins (e.g. transcription factors) for their corresponding specific sites. Thus it is quite possible that y3BP1 would be capable of binding Pu motif triplexes, should they existin vivo. Though yeast 3BP1 may have a high affinity for Pu motif triplexes, it is possible this structure is not the true target of this proteinin vivo. To better understand the binding specificity of y3BP1, competition experiments were undertaken with a variety of different DNA structures. These included several quadruplex DNAs (both parallel tetraplexes and antiparallel hairpin dimers and intramolecular quadruplexes), triplex DNAs (both Pu motif and Py motif), duplex DNAs (both A/T- and G/C-rich), and the single-stranded TFO ODN 1. Their sequences and structures are shown in Fig. 1. Binding reactions were modified in some cases to maintain the integrity of these DNA structures. For example, competition experiments with single-stranded ODN 1 were performed in buffer A containing HEPES-Li+ and LiCl instead of HEPES-Na+ and KCl, respectively, to minimize formation of G4-containing species with this G-ric
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