Sequence-specific Binding Protein of Single-stranded and Unimolecular Quadruplex Telomeric DNA from Rat Hepatocytes
1997; Elsevier BV; Volume: 272; Issue: 25 Linguagem: Inglês
10.1074/jbc.272.25.15881
ISSN1083-351X
Autores Tópico(s)DNA and Nucleic Acid Chemistry
ResumoA rat liver nuclear protein, unimolecular quadruplex telomere-binding protein 25, (uqTBP25) is described that binds tightly and specifically single-stranded and unimolecular tetraplex forms of the vertebrate telomeric DNA sequence 5′-d(TTAGGG) n -3′. A near homogeneous uqTBP25 was purified by ammonium sulfate precipitation, chromatographic separation from other DNA binding proteins, and three steps of column chromatography. SDS-polyacrylamide gel electrophoresis and Superdex© 200 gel filtration disclosed for uqTBP25 subunit and nativeM r values of 25.4 ± 0.5 and 25.0 kDa, respectively. Sequences of uqTBP25 tryptic peptides were closely homologous, but not identical, to heterogeneous nuclear ribonucleoprotein A1, heterogeneous nuclear ribonucleoprotein A2/B1, and single-stranded DNA-binding proteins UP1 and HDP-1. Complexes of uqTBP25 with single-stranded or unimolecular quadruplex 5′-d(TTAGGG)4-3′, respectively, had dissociation constants,K d, of 2.2 or 13.4 nm. Relative to d(TTAGGG)4, complexes with 5′-r(UUAGGG)4-3′, blunt-ended duplex telomeric DNA, or quadruplex telomeric DNA had >10 to >250-fold higher K d values. Single base alterations within the d(TTAGGG) repeat increased theK d of complexes with uqTBP25 by 9–215-fold. Association with uqTBP25 protected d(TTAGGG)4 against nuclease digestion, suggesting a potential role for the protein in telomeric DNA transactions. A rat liver nuclear protein, unimolecular quadruplex telomere-binding protein 25, (uqTBP25) is described that binds tightly and specifically single-stranded and unimolecular tetraplex forms of the vertebrate telomeric DNA sequence 5′-d(TTAGGG) n -3′. A near homogeneous uqTBP25 was purified by ammonium sulfate precipitation, chromatographic separation from other DNA binding proteins, and three steps of column chromatography. SDS-polyacrylamide gel electrophoresis and Superdex© 200 gel filtration disclosed for uqTBP25 subunit and nativeM r values of 25.4 ± 0.5 and 25.0 kDa, respectively. Sequences of uqTBP25 tryptic peptides were closely homologous, but not identical, to heterogeneous nuclear ribonucleoprotein A1, heterogeneous nuclear ribonucleoprotein A2/B1, and single-stranded DNA-binding proteins UP1 and HDP-1. Complexes of uqTBP25 with single-stranded or unimolecular quadruplex 5′-d(TTAGGG)4-3′, respectively, had dissociation constants,K d, of 2.2 or 13.4 nm. Relative to d(TTAGGG)4, complexes with 5′-r(UUAGGG)4-3′, blunt-ended duplex telomeric DNA, or quadruplex telomeric DNA had >10 to >250-fold higher K d values. Single base alterations within the d(TTAGGG) repeat increased theK d of complexes with uqTBP25 by 9–215-fold. Association with uqTBP25 protected d(TTAGGG)4 against nuclease digestion, suggesting a potential role for the protein in telomeric DNA transactions. Linear eukaryotic chromosomes end with a specialized DNA-protein structure termed the telomere that guards the chromosome terminus against degradative attack or fusion with ends of other chromosomes (1Blackburn E.H. Cell. 1994; 77: 621-623Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 2Zakian V.A. Science. 1995; 270: 1601-1607Crossref PubMed Scopus (796) Google Scholar, 3Greider C.W. Annu. Rev. Biochem. 1996; 65: 337-365Crossref PubMed Scopus (912) Google Scholar, 4Lundblad V. Wright W.E. Cell. 1996; 87: 369-375Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Telomeric DNA consists of evolutionarily conserved short, tandemly repeated nucleotide sequences. The telomeric DNA strand, oriented 5′ to 3′ toward the chromosome end (“G-strand”) in all vertebrates, slime molds, filamentous fungi, andTrypanosoma, is a repeated 5′-d(TTAGGG)-3′ sequence paired to a complementary 5′-d(CCCTAA)-3′ strand. 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In this work we describe the purification from rat hepatocytes and characterization of a 25-kDa monomeric protein, termed uqTBP25, that binds tightly and in a sequence-specific fashion single-stranded and unimolecular tetraplex forms of the G-strand of telomeric DNA. A partial amino acid sequence of uqTBP25 is closely homologous but not identical with sequences of hnRNP 1The abbreviations used are: hnRNP, heterogenous nuclear ribonucleoprotein; HPLC, high pressure liquid chromatography; DTT, dithiothreitol; MalNEt, N-ethylmaleimide; TEMED,N,N,N′,N′-tetramethylethylenediamine; PAGE, polyacrylamide gel electrophoresis. A1 and hnRNP A2/B1 and their respective derivative single-stranded DNA-binding proteins UP1 and HDP-1. Protein uqTBP25 is distinguished from hnRNP A1 and A2/B1 by its molecular size, preferential binding to DNA over RNA, and sequence-specific binding to the telomeric DNA G-strand. This protein also differs from UP1 and HDP-1 by its selective binding to the G-strand of telomeric DNA and by its failure to significantly stimulate the activity of DNA polymerase α. Radioactively 5′-labeled [γ-32P]ATP (∼3000 Ci/mmol), [α-32P]dGTP (∼3000 Ci/mmol), Klenow fragment ofEscherichia coli polymerase I, and molecular mass Rainbow™ marker proteins were products of Amersham Corp. Synthetic DNA oligomers, listed in Table I, were purchased from Operon Technologies. The HPLC-purified RNA oligomer r(UUAGGG)4 (TableI) was a product of Midland Reagent. Boric acid, β-mercaptoethanol, dithiothreitol (DTT), N-ethylmaleimide (MalNEt), poly(dG)·poly(dC), thymidine 3′,5′-diphosphate, dimethyl sulfate, leupeptin, aprotinin, benzamidine, phenylmethylsulfonyl fluoride, Nonidet P-40, Sephadex G-50, phenyl-Sepharose, proteinase K, salmon sperm DNA, soybean trypsin inhibitor, and micrococcal nuclease were supplied by Sigma. DEAE-cellulose (DE-52) and DE-81 filter paper were the products of Whatman. Total RNA from yeast was supplied by Boehringer Mannheim. Bacteriophage T4 polynucleotide kinase and RNasin were provided by Promega. Acryl/bisacrylamide (19:1 or 30:1.2) was purchased from Amresco. Bacteriophage T4 gene 32 protein was purchased from Boehringer Mannheim. Immunoaffinity-purified calf thymus DNA polymerase α was the gift of Dr. L. A. Loeb (University of Washington). Kodak XAR5 and Biomax MR-1 autoradiographic film, urea, TEMED, bromphenol blue, and xylene cyanol FF were supplied by IBI. HiTrap Blue HPLC column and Superdex© 200 HPLC gel filtration column were provided by Pharmacia Biotech Inc. Econo-Pac S HPLC cartridge, reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and molecular weight protein standards were the products of Bio-Rad. Novex provided Multimark™ molecular size protein standards. Biotrace polyvinylidene difluoride binding matrix membranes were supplied by Gelman Sciences. Full-length DNA oligomers were purified by electrophoresis through a 8 m urea, 15% polyacrylamide denaturing gel (acryl/bisacrylamide, 19:1) as we described (47Fry M. Perrino F.W. Levy A. Loeb L.A. Nucleic Acids Res. 1988; 16: 199-211Crossref PubMed Scopus (11) Google Scholar). The purified DNA or RNA oligomers were labeled at their 5′-end with32P in a bacteriophage T4 polynucleotide kinase-catalyzed reaction (48Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Crossref PubMed Scopus (130) Google Scholar). Oligomers were maintained in their single-stranded conformation as a 0.25–0.70 μm solution in 1.0 mm EDTA, 10 mm Tris-HCl buffer, pH 8.0 (TE buffer), and were boiled immediately prior to use. Double-stranded telomeric DNA was prepared by annealing a 1.25-fold molar excess of a cytosine-rich sequence with a complementary guanine-rich oligomer, and duplex DNA molecules were electrophoretically resolved from residual DNA single strands as we described (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Labeling of a protruding end of the annealed duplex DNA was catalyzed by the Klenow fragment ofE. coli polymerase I using 5′-[α-32P]dGTP as we described previously (47Fry M. Perrino F.W. Levy A. Loeb L.A. Nucleic Acids Res. 1988; 16: 199-211Crossref PubMed Scopus (11) Google Scholar). Unimolecular (G′4) and bimolecular (G′2) tetraplex forms of TeR DNA were prepared, their stoichiometry was verified, and their stabilization by Hoogsteen bonds was demonstrated, as we described in detail elsewhere (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Parallel G4 quadruplex forms of oligomers Q and single Q were prepared according to Sen and Gilbert (48Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Crossref PubMed Scopus (130) Google Scholar), and their stoichiometry was shown to be tetramolecular as recently described (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The parallel quadruplex form of d(CGG)8 was prepared, and its structure was verified as previously detailed (49Fry M. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4950-4954Crossref PubMed Scopus (318) Google Scholar). The DNA binding activity of uqTBP25 was monitored by electrophoretic mobility shift assay as we described previously (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,50Weisman-Shomer P. Fry M. J. Biol. Chem. 1993; 268: 3306-3312Abstract Full Text PDF PubMed Google Scholar). In a typical assay for the binding of single-stranded TeR-4 or TeR-2 DNA, 5.0–15.0 ng of 32P-5′-labeled TeR-2 or TeR-4 DNA was incubated at 4 °C for 20 min with 30–3000 ng of purified or crude protein fraction in a 15-μl final volume of buffer D (0.5 mm EDTA, 20% glycerol in 25 mm Tris-HCl buffer, pH 7.5). The binding mixture was electrophoresed in a Mini PROTEAN II electrophoresis system (Bio-Rad) at 4 °C under 10 V/cm through a nondenaturing 6% polyacrylamide gel (acryl/bisacrylamide, 30:1.2) in 0.6 × TBE buffer (1.2 mm EDTA in 0.54m Tris borate buffer, pH 8.3) until a bromphenol blue tracking dye migrated 2.5–4.0 cm into the gel. The gels were dried on DE-81 filter paper and exposed to x-ray film or to a phosphor imaging plate (Fuji). The proportion of free and uqTBP25-bound TeR DNA was determined by phosphor imaging, and their amounts were deduced from the known specific activity of the labeled DNA probe. One unit of uqTBP25 DNA binding activity was defined as the amount of uqTBP25 that bound 66 pmol of single-stranded TeR-2 DNA under the described standard conditions. Standard assay conditions were also employed for the binding of tetramolecular G4 quadruplex DNA and of double-stranded DNA. Binding of tetraplex G′2 TeR DNA or G′4 TeR DNA was assayed as described above except that 50 mm KCl or 50 mmNaCl, respectively, was added to the DNA binding mixture to preserve the quadruplex structure of the DNA, the 0.6 × TBE gel running buffer contained 50 mm KCl or 50 mm NaCl as necessary, and electrophoresis was performed at 4 °C. SDS-PAGE and silver or Coomassie Blue staining of resolved protein bands was carried out as we described previously (50Weisman-Shomer P. Fry M. J. Biol. Chem. 1993; 268: 3306-3312Abstract Full Text PDF PubMed Google Scholar). Molecular size protein markers were the Amersham Rainbow™, Novex Multimark™, or Bio-Rad prestained or unstained molecular weight standards. Southwestern analysis was conducted according to Petracek et al. (31Petracek M.E. Konkel L.M.C. Kable M.L. Berman J. EMBO J. 1994; 13: 3648-3658Crossref PubMed Scopus (45) Google Scholar) with the minor modifications that we recently introduced (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). TeR-4 DNA binding activity was detected in nuclear extracts by exposing the electrophoretically resolved proteins to 0.85 μg of32P-5′-labeled TeR-4 DNA in the presence of 50 mm NaCl. In a typical preparation, protein uqTBP25 was purified to near homogeneity from ∼700 g of liver tissue from adult rats. Salt extracts of nonhistone nuclear proteins were prepared from isolated hepatocyte nuclei as we described elsewhere (52Sharf R. Weisman-Shomer P. Fry M. Biochemistry. 1988; 27: 2990-2997Crossref PubMed Scopus (9) Google Scholar), except that the composition of the extraction buffer was 0.4m NaCl, 1 mm phenylmethylsulfonyl fluoride, 0.1 mm benzamidine, and 10 μg/ml each of the protease inhibitors soybean trypsin inhibitor, leupeptin, and aprotinin in buffer D. The preparation of protein extracts and all of the subsequent steps of uqTBP25 purification were conducted at 4 °C. Electrophoretic mobility shift assays and Southwestern analysis showed that rat liver nuclear extracts contained G′4 TeR-4 DNA binding activity with an approximate molecular size of 24 kDa (see “Results”). This protein was further purified by successive steps of ammonium sulfate precipitation and column chromatography. Elution profiles of the DNA binding activity from the various columns, as assessed by electrophoretic mobility shift analysis were identical when32P-5′-labeled single-stranded TeR-2 or TeR-4 DNA or unimolecular tetraplex G′4 TeR-4 DNA were used as probes. In an initial purification step, extract proteins were precipitated by 50% ammonium sulfate and removed by centrifugation. The majority of the TeR DNA binding activity that remained in the supernatant was subsequently precipitated by 70% ammonium sulfate. The 70% ammonium sulfate precipitate was resuspended in buffer D and was dialyzed overnight at 4 °C against ∼200 volumes of the same buffer. After adding NaCl to the dialyzed protein fraction to a final concentration of 50 mm, it was mixed with heat-denatured salmon sperm DNA and incubated for 20 min at 4 °C at a protein:DNA ratio of 30:1 (w/w). Whereas the major TeR-binding protein qTBP42 (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and additional single-stranded DNA-binding proteins associated tightly with the denatured DNA, uqTBP25 did not bind detectably to it. The strong binding of DNA and DNA-protein complexes to DEAE-cellulose (53Fry M. Lapidot J. Weisman-Shomer P. Biochemistry. 1985; 24: 7549-7556Crossref PubMed Scopus (8) Google Scholar) was subsequently utilized to separate uqTBP25 from qTBP42 (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and from additional proteins that associated with denatured DNA. The protein-DNA mixture was loaded at a ratio of 4.0 mg of protein/ml of packed resin, onto a DE-52 column equilibrated in buffer D containing 50 mm NaCl. The protein-loaded column was washed with 0.5 and subsequently with 1.5 packed column volumes of the equilibration buffer, and bound proteins were eluted by two packed column volumes each of 100 and 225 mm NaCl in buffer D. Electrophoretic mobility shift analysis and SDS-PAGE separation of UV cross-linked TeR-4 DNA-protein complexes, respectively, revealed TeR-4 DNA binding activity and UV cross-linked TeR-4 DNA-protein complexes of 34 kDa in the 50 mm NaCl wash fractions. Similar analysis did not reveal DNA-protein complexes in the 100 mm NaCl fraction; instead, multiple complex bands, the dominant of which was qTBP42 (33Sarig G. Weisman-Shomer P. Erlitzki R. Fry M. J. Biol. Chem. 1997; 272: 4474-4482Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), were present in the 225 mm NaCl eluate. The 50 mm NaCl eluate fractions were pooled together and dialyzed overnight against ∼50 volumes of buffer P (0.5 mm EDTA, 20% glycerol in 25 mm NaPO4 buffer, pH 7.0). The dialyzed fractions were loaded at a ratio of 24.0 mg of protein/ml of packed resin onto a 5.0-ml Econo-Pac S column equilibrated in buffer P and mounted on a GradiFrac low pressure chromatography device (Pharmacia). The loaded column was washed with 7.5 packed resin volumes of buffer P, and bound proteins were eluted from the column by a linear gradient of 19.5 column volumes of 0.0–1.0 m NaCl in buffer P. Fifty fractions were collected, and as done in every subsequent chromatography, aliquots were dialyzed overnight against 150 volumes of buffer D and then assayed for G′4 TeR-4 DNA binding. G′4 TeR-4 DNA binding activity of uqTBP25 was detected in the 150–320 mm NaCl eluate both by electrophoretic mobility shift analysis and by the identification in SDS-PAGE of a ∼34-kDa UV-cross-linked protein-TeR-4 DNA complex. The active fractions were pooled together, dialyzed overnight against 150 volumes of P2 buffer (0.5 mm EDTA, 20% glycerol in 10 mmNaPO4, pH 7.0), and loaded at a ratio of 8.5 mg of protein/ml of packed resin onto a 1.0-ml HiTrap Blue HPLC column equilibrated in P2 buffer and mounted on a GradiFrac device. The loaded column was washed by six column volumes of the equilibration buffer, and adsorbed proteins were eluted by a 21-packed column volume linear gradient of 0.0–4.0 m NaCl in P2 buffer. Fifty fractions were collected, aliquots were dialyzed, and uqTBP25 binding activity was detected by electrophoretic mobility shift analysis and SDS-PAGE of UV cross-linked protein-TeR-4 DNA complexes in fractions that were eluted from HiTrap Blue by 2.5–3.5 m NaCl. Fractions containing the binding activity were pooled together and dialyzed overnight against ∼50 volumes of 4.0 m NaCl in buffer S (1.0 mm EDTA in 25 mm Tris-HCl buffer, pH 7.5) and loaded at a ratio of 1.0 mg of protein:1.0 ml of packed resin onto a phenyl-Sepharose column equilibrated in buffer S. The loaded column was washed with two packed column volumes of the equilibration buffer, and bound proteins were eluted by a stepwise gradient of 4.0–0.0m NaCl in buffer S followed by a 40% ethylene glycol wash to elute proteins that remained adsorbed to phenyl-Sepharose at 0.0m NaCl. Fractions were collected into Nonidet P-40 (0.05% final concentration), and the activity of uqTBP25 was detected in fractions that were eluted from the phenyl-Sepharose column by 1.0–0.5m NaCl. Silver and Coomassie Blue staining of the eluted proteins indicated that a 25-kDa species was the major protein eluted by 1.0–0.5 m NaCl (Fig. 2 C), whereas the majority of the proteins that were loaded onto the column were eluted by 40% ethylene glycol. Determination of the protein content of the collected fractions and silver or Coomassie Blue staining of SDS-PAGE-resolved protein bands was directly performed on fractions that were dialyzed against water. Fractions that were used for the assay of DNA binding activity were stabilized by the immediate addition soybean trypsin inhibitor protein (200 μg/ml final concentration), and following their dialysis overnight against ∼200 volumes of buffer D, they were stored in aliquots at −80 °C. Under these storage conditions, the DNA binding activity was fully preserved for at least 4 months.Table IDNA and RNA oligomers used in this studyOligomer designationLengthNucleotide sequenceTeR-51-aClusters of guanine residues are underlined.38-mer5′-GTCGACCCGGGTTAGGGTTAGGGTTAGGGTTAGGGTTA)-3′TeR-41-aClusters of guanine residues are underlined.24-mer5′-d(TTAGGGTTAGGGTTAGGGTTAGGG)-3′TeR-31-aClusters of guanine residues are underlined.23-mer5′-d(GTCGACCCGGGTTAGGGTTAGGG)-3′TeR-21-aClusters of guanine residues are underlined.23-mer5′-d(TAGACATGTTAGGGTTAGGGTTA)-3′TeR-11-aClusters of guanine residues are underlined.17-mer5′-d(TAGACATGTTAGGGTTA)-3′rTeR-41-aClusters of guanine residues are underlined.24-mer5′-r(UUAGGGUUAGGGUUAGGGUUAGGG)-3′TeR-4 C1-bClusters of cytosine residues are underlined.24-mer5′-d(CCCTAACCCTAACCCTTACCCTTA)-3′TeR-3 C1-bClusters of cytosine residues are underlined.23-mer5′-d(CCCTAACCCTAACCCGGGTCGAC)-3′TeT-41-aClusters of guanine residues are underlined.,1-cLoci of a substituted nucleotide relative to TeR-4 DNA are doubly underlined.24-mer5′-d(GGGGTTGGGGTTGGGGTTGGGGTT)-3′TeT-21-aClusters of guanine residues are underlined.,1-cLoci of a substituted nucleotide relative to TeR-4 DNA are doubly underlined.23-mer5′-d(TAGACATGTTGGGGTTGGGGTTG)-3′Mut1 TeR-41-cLoci of a substituted nucleotide relative to TeR-4 DNA are doubly underlined.24-mer5′-d(TTAGAGTTAGAGTTAGAGTTAGAG)-3′Mut2 TeR-41-cLoci of a substituted nucleotide relative to TeR-4 DNA are doubly underlined.24-mer5′-d(TAAGGGTAAGGGTAAGGGTAAGGG)-3′Hook TeR-41-aClusters of guanine residues are underlined.32-mer5′-d(CTGGACCCGGGTTAGGGTTAGGGTTAGGGTTA)-3′Hook TeR-4 C1-bClusters of cytosine residues are underlined.29-mer5′-d(TAACCCTAACCCTAACCCTAACCCGGGTC)-3′Q1-aClusters of guanine residues are underlined.20-mer5′-d(TACAGGGGAGCTGGGGTAGA)-3′Single Q1-aClusters of guanin
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