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• W2018172829 abstract "G4 nucleic acids are quadruplex structures involving guanine-rich sequences that form in vitro under moderate conditions. Experimental evidence exists supporting biological functions for these elements; however, direct demonstration of G4 nucleic acids in vivo has not yet been achieved. Here we purify and characterize a yeast protein, G4p2, which has a specific affinity for G4 nucleic acids. G4p2 binds equivalently to RNA and DNA in G4 form. The Keqfor G4p2 binding to a G4 DNA oligomer is 2.2 × 108M−1under near physiological conditions. We have cloned and sequenced the gene encoding G4p2 and have shown it to be identical to MPT4 and STO1. MPT4 was isolated in a screen for multicopy suppressors of staurosporine sensitivity in POP2 cells. Pop2 is a complex regulatory factor that participates, in part, in the repression of certain genes in the absence of glucose (Sakai, A., Chibazakura, T., Shimizu, Y., and Hishinuma, F. (1992) Nucleic Acids Res. 20, 6227-6233). STO1 was isolated as a multicopy suppressor of TOM1, an uncharacterized mutation that leads to temperature-sensitive cell cycle arrest at the G2/M boundary. Suppression of these mutations by G4p2 indicate this G4 nucleic acid binding protein may function in signal transduction pathways regulated by protein kinases, which control carbon source utilization, and in cell cycle progression. G4 nucleic acids are quadruplex structures involving guanine-rich sequences that form in vitro under moderate conditions. Experimental evidence exists supporting biological functions for these elements; however, direct demonstration of G4 nucleic acids in vivo has not yet been achieved. Here we purify and characterize a yeast protein, G4p2, which has a specific affinity for G4 nucleic acids. G4p2 binds equivalently to RNA and DNA in G4 form. The Keqfor G4p2 binding to a G4 DNA oligomer is 2.2 × 108M−1under near physiological conditions. We have cloned and sequenced the gene encoding G4p2 and have shown it to be identical to MPT4 and STO1. MPT4 was isolated in a screen for multicopy suppressors of staurosporine sensitivity in POP2 cells. Pop2 is a complex regulatory factor that participates, in part, in the repression of certain genes in the absence of glucose (Sakai, A., Chibazakura, T., Shimizu, Y., and Hishinuma, F. (1992) Nucleic Acids Res. 20, 6227-6233). STO1 was isolated as a multicopy suppressor of TOM1, an uncharacterized mutation that leads to temperature-sensitive cell cycle arrest at the G2/M boundary. Suppression of these mutations by G4p2 indicate this G4 nucleic acid binding protein may function in signal transduction pathways regulated by protein kinases, which control carbon source utilization, and in cell cycle progression. INTRODUCTIONDNA and RNA containing guanine tracts will associate in vitro in the presence of salt into four-stranded right-handed helices stabilized by a guanine base quartet (for review see Refs. 1Guschlbauer W. Chantof J.F. Thiele D. J. Biomol. Struct. & Dyn. 1990; 8: 491-511Google Scholar, 2Sen D. Gilbert W. Curr. Opin. Struct. Biol. 1991; 1: 435-438Google Scholar, 3Sundquist W.I. Nucleic Acids Mol. Biol. 1991; 5: 1-24Google Scholar, 4Williamson J.R. Curr. Opin. Struct. Biol. 1993; 3: 357-362Google Scholar, 5Williamson J.R. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 703-730Google Scholar). Three major helical isoforms, characterized by parallel or cis or trans antiparallel strand orientations, have been identified by NMR and x-ray studies. These quadruplex structures are called the G4 nucleic acids. G4 structures anneal at moderate to low rates under physiological conditions and are greatly stabilized by millimolar concentrations of potassium, which is efficiently chelated by guanine carbonyl oxygens that line the axial cavity of the helix.Do G4 structures arise in vivo? Presently, this question is unresolved, although there is tantalizing evidence supporting roles for G4 structures at eukaryotic telomeres (7Sundquist W.I. Klug A. Nature. 1989; 342: 825-829Google Scholar, 8Williamson J.R. Raghuraman M.K. Cech T.R. Cell. 1989; 59: 871-880Google Scholar) and at retroviral dimer linkage sequences (9Sundquist W.I. Heaphy S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3393-3397Google Scholar, 10Awang G. Sen D. Biochemistry. 1993; 32: 11453-11457Google Scholar). Also, Sep1/Dst2/Xrn1/Kem1 is a yeast enzyme that catalyzes DNA strand exchange (11Tishkoff D.X. Johnson A.W. Kolodner R.D. Mol. Cell. Biol. 1991; 11: 2593-2608Google Scholar, 12Dykstra C.C. Kitada K. Clark A.B. Hamatake R.K. Sugino A. Mol. Cell. Biol. 1991; 11: 2583-2592Google Scholar), hydrolyzes RNA (13Larimer F.W. Stevens A. Gene (Amst.). 1990; 95: 85-90Google Scholar), and possesses a nucleolytic activity specific for DNA oligomers containing G4 domains (14Liu Z. Gilbert W. Cell. 1994; 77: 1083-1092Google Scholar). Null alleles of this gene lead to mitotic loss of chromosomes and meiotic arrest at pachytene (15Kim J. Ljungdahl P.O. Fink G.R. Genetics. 1990; 126: 799-812Google Scholar). These defects may be due to an inability of the mutant cell to properly process G4 structures that may arise during the course of chromosomal replication, recombination, or alignment. Further, a G4 DNA-annealing activity capable of accelerating the conversion of oligomers into G4 structures by a factor of 105was identified in a subunit of a Oxytricha telomere binding complex, suggesting that G4 structures do exist, perhaps transiently, at chromosome ends (16Fang G. Cech T.R. Cell. 1993; 74: 875-885Google Scholar).To explore the possible biological functions of G4 nucleic acids, we have probed extracts for activities specific for G4 DNA. We have identified and characterized a yeast protein, G4p2, that displays an avid and specific binding activity for G4 nucleic acids. G4p2 slightly prefers substrates containing antiparallel G4 domains over those containing the parallel form and strongly prefers substrates containing multiple G4 domains. G4p2 binds equivalently to DNA and RNA in G4 form. The protein was purified, and the gene encoding G4p2 was isolated and sequenced. The G4P2 gene is identical to MPT4 and STO1, genes that appear to function in a protein kinase-controlled signal transduction pathway and in cell cycle progression, respectively.EXPERIMENTAL PROCEDURESPreparation of G4 OligomersDNA and RNA oligomers (see Fig. 1) were synthesized on an ABI model 392 DNA synthesizer, and full-length products were purified on 8 M urea/polyacrylamide gels. G4 structures were annealed and purified on native polyacrylamide gels containing KCl under conditions specified by Sen and Gilbert (17Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Google Scholar) and stored in 10 m M Tris, pH 7.6, 1 m M EDTA, 100 m M KCl at 4°C. Yields were determined by absorbance at 260 nm.Mobility Shift Assays, Competitions, and SDS-PAGEGL(G4) (see Fig. 1) was 5′-labeled with [γ-32P]ATP and T4 polynucleotide kinase and precipitated twice with ethanol. 5000 counts per second of labeled GL(G4) (about 8 fmol or 320 pg, unless otherwise indicated) was added to extract or G4p2 fractions in binding buffer (20 m M NaHepes, pH 7.3, 1 m M EDTA, 50 m M KCl, 5 m M BME,1 1The abbreviations used are: BMEβ-mercaptoethanolPAGEpolyacrylamide gel electrophoresis. 5 m M NaPO4, 0.05% Triton X-100, 2.5% glycerol, 1 μg of bovine serum albumin, 2.5 μg of double-stranded salmon sperm DNA, tracking dyes) in a final volume of 10 μl and incubated at room temperature for 10 min. The reactions were loaded on 5% polyacrylamide gels and run at room temperature in 50 m M TBE (50 m M Tris borate, 1 m M EDTA, pH 8.2) with 1 m M KCl at 12.5 V/cm. The gels were dried and exposed for several hours to x-ray film or to an imaging plate (Fuji) for 15 min.Competition experiments were carried out as above, with saturating amounts of GL(G4) probe. 10-fold serial dilutions of competitor nucleic acids were premixed with extract or G4p2 fractions in binding buffer just prior to adding the probe. Relative mass amounts of competitor to probe ranged from 0.1 to 1000 and were determined by absorbance at 260 nm. Single-stranded competitors were fully denatured by treatment with 100 m M NaOH at 100°C just prior to use.SDS-PAGE was carried out by standard procedures (18Gallagher S.R. Smith J.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Vol. 2. John Wiley & Sons, New York1992: 10.2.1-10.2.21Google Scholar). 10-μl aliquots of G4p2 fractions were loaded on 10% SDS-PAGE gels, and protein bands were visualized by staining twice with a silver staining reagent (Bio-Rad).Large Scale Preparation of Yeast ExtractDiploid yeast (Saccharomyces cerevisiae, strain SK1) was cultured in 50 liters of YPD (1% yeast extract, 2% peptone, 1% dextrose), harvested at midlog phase, and lysed in liquid nitrogen according to published procedures (19Sorger P.K. Ammerer G. Shore D. Creighton T.E. Protein Function: A Practical Approach. IRL Press, Oxford1989: 199-223Google Scholar). The extract was prepared at 4°C. Proteins were extracted from lysed cells in extraction buffer (20 m M NaHepes, pH 7.3, 1 m M EDTA, 10 m M MgCl2, 420 m M KCl, 0.05% Triton X-100, 5 m M BME, 10% glycerol, 1 m M phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A) for 1 h at 0.5 g of cell paste/ml. The extract was cleared by centrifugation at 25,000 × g for 15 min and dialyzed (Spectra/Por 7; molecular weight cutoff, 3500) with one change for 48 h against 10 volumes of dialysis buffer (20 m M NaHepes, pH 7.3, 1 m M EDTA, 45 m M KCl, 0.05% Triton X-100, 5 m M BME, 10% glycerol). The dialysate was cleared again by centrifugation at 90,000 × g for 1 h and frozen at −100°C. The protein concentration and yield were determined to be 25 mg/ml and 0.07 g of protein/g of cell paste, respectively, using a Bradford dye binding assay (Bio-Rad).Purification of G4p2All extract and G4p2 fraction manipulations were done at 4°C. Thawed yeast extract (12.5 ml) was dialyzed (Spectra/Por 7; molecular weight cutoff, 3500) against 1250 ml of buffer A (20 m M sodium bicarbonate, pH 11, 1 m M EDTA, 50 m M KCl, 100 m M NaCl, 0.05% Triton X-100, 5 m M BME, 10% glycerol) for 12 h and cleared by centrifugation at 10,000 × g for 10 min. The supernatant (240 mg in 12.5 ml) was loaded at 25 ml/h on a 25-ml S-Sepharose FF (Pharmacia) bed, which had been equilibrated in buffer A and packed in a 2.5 × 10-cm EconoColumn (Bio-Rad). The bed was washed with 125 ml of buffer A, and proteins were eluted on simultaneous 50-ml linear gradients of decreasing NaCl concentration (100-0 m M) and increasing NaPO4pH 11 concentration (0-100 m M). Forty 1.25-ml fractions were collected and analyzed by mobility shift assay for G4p2 and by SDS-PAGE. Fractions 17-25 (11.25 ml) containing partially purified G4p2 were pooled.Half of the partially purified G4p2 from the previous step (6 ml) was concentrated with Microcon-10 microconcentrators (Amicon) to 0.15 ml, washed twice with 7 volumes of buffer B (20 m M NaHepes, pH 7.3, 1 m M EDTA, 50 m M KCl, 0.05% Triton X-100, 5 m M BME, 10% glycerol), and recovered in a volume of 0.5 ml. The sample was made 100 m M NaOH for 5 min and neutralized with HCl.A 1-ml G4 DNA affinity bed was prepared as follows. Oligomer GL (see Fig. 1) with biotin incorporated next to the 3′ end (Biotin-ON phosphoramidite, Clontech) was synthesized at a 1-μmol scale. The unpurified oligomer was converted into parallel G4 form under conditions specified by Sen and Gilbert (17Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Google Scholar). An aliquot was labeled with [γ-32P]ATP and T4 polynucleotide kinase and shown by gel electrophoresis to be composed of parallel G4 DNA and higher order G4 structures (20Sen D. Gilbert W. Biochemistry. 1992; 31: 65-70Google Scholar). The G4 DNA (3.95 mg) was bound to 2 ml of 50% streptavidin-agarose (Sigma) in 10 m M Tris, pH 7.6, 1 m M EDTA, 100 m M KCl by gentle rocking for 30 min. The affinity matrix was washed twice in buffer B with 100 m M NaCl, packed in a 0.7 × 5-cm EconoColumn (Bio-Rad), and equilibrated at 4°C.Partially purified G4p2 from the previous step (0.5 ml) was loaded at 2.3 ml/h on the G4 DNA affinity column and washed with 5 ml of buffer B with 100 m M NaCl, and bound proteins were eluted with a 5-ml convex exponential gradient of increasing NaCl concentration (100 m M to 1 M). Fifty 0.1-ml fractions were collected and assayed by mobility shift assay for G4p2 and by SDS-PAGE (see Fig. 3). Fractions 21-27 (0.7 ml) containing largely pure G4p2 were pooled.Figure 3:Purification of G4p2. A, fractions from the G4 DNA affinity column were analyzed by mobility shift assay with 320 pg of probe as described under “Experimental Procedures.” 1 μl of a 100-fold dilution of each fraction was analyzed. The input fraction (IN) is pooled, and concentrated fractions 17 through 25 were obtained from the S-Sepharose FF pH 11 column. G4p2 is indicated. B, SDS-PAGE of the same fractions as in A. In each case, 10 μl of undiluted fraction was analyzed. G4p2 and molecular weights of markers are indicated. The gel was stained twice with silver.View Large Image Figure ViewerDownload (PPT)Determination of the Keq for G4p2 Binding to G4 DNAMobility shift experiments as described above were carried out with 2.7 fm of GL(G4) probe (Fig. 1) and with 4-fold serial dilutions of G4p2 (1350, 338, 84.4, 21.1, and 5.27 fm) obtained from the G4 DNA affinity column (prep 2; see “Results”). Stock G4p2 was quantitated by titrating a dilution of G4p2 with increasing amounts of precisely quantitated GL(G4) DNA probe until binding to G4p2 was saturated. At saturation, molarity of the bound probe is equivalent to that of G4p2. By this method, prep 2 contains 1.35 μM G4p2. The dried gels were exposed to an imaging plate (Fuji), and the amounts of free probe at each G4p2 dilution were measured. Keqwas estimated from a plot of log(free G4p2 concentration) versus log(bound probe = (total probe - free probe)/free probe) (21Fried M.G. Crothers D.M. J. Mol. Biol. 1984; 172: 241-262Google Scholar). The free G4p2 concentration was estimated by subtracting the concentration of bound probe at each dilution, which equals the concentration of bound G4p2, from that of input G4p2.Peptide Sequencing and DNA Hybridization ProbesPartially purified G4p2 (2.7 ml) obtained from the S-Sepharose column was precipitated with 12.5% trichloroacetic acid and 0.5 mg/ml deoxycholate and fractionated on a 10% SDS-PAGE gel. The proteins were electrotransferred to a nitrocellulose membrane and visualized by staining with 0.1% Ponceau S in 5% acetic acid. G4p2 was excised and sequenced (Harvard Microchemistry Facility). The sequences of two internal tryptic peptides (NT-49, TAQLSLQDYLNQQANNQFNKVPEAK and NT-40, EAQADAAAEIAEDAAEAEDAGKPK) were obtained. A unique oligomer (49C, 5′-CAAGATTACTTGAACCAACAAGCTAACAACCAATTCAACAAGGTTCCAGAAGCTAA-3′) incorporating most frequent codon usage information for yeast (22Davis T.N. Thorner J. Methods Enzymol. 1987; 139: 248-262Google Scholar) was synthesized based on reverse translated NT-49. This hybridization probe was tested on Southern blots of yeast genomic DNA and was found suitable for library screening.Isolation, Sequencing, and Subcloning of the G4p2 GeneOligomer 49C labeled with [γ-32P]ATP and T4 polynucleotide kinase was used to screen 6 × 103plaques of recombinant λEMBL3a containing a yeast genomic library (23Snyder M. Elledge S. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 730-734Google Scholar). Five positive plaques were pooled and rescreened at low density. Three positive plaques from the second screen were selected for further analysis.Selected phage DNA was pooled and used as template for thermal cycle sequencing with the CircumVent (New England Biolabs) sequencing reagents. To obtain access to the G4p2 gene, 1.2 pmol of a 128-fold degenerate oligomer (49A, 5′-AA(C/T)CA(A/G)CA(A/G)GC(A/G/C/T)AA(C/T)AA(C/T)CA-3′) derived from reverse translated NT-49 was labeled with [γ-32P]ATP and T4 polynucleotide kinase and was used as a primer on 400 ng of template DNA. The nucleotide sequence obtained permitted the synthesis of unique primers to extend similarly and to finish both strands of the DNA sequence encoding G4p2. A 5-kilobase EcoRI fragment from a λEMBL3a clone containing the intact G4p2 coding sequence was inserted into pUC19 using standard methods, generating the subclone pUC19G4p2.Preparation of Recombinant G4p2A bacterially expressed G4p2 fusion to glutathione S-transferase was produced with the pGEX-3X (Pharmacia) vector system. Two complementary oligomers (top, 5′-GATCTCCAACCCATTTGATTTGTTAGGTAACGACGTCGTCG-3′ and bottom, 5′-GATCCGACGACGTCGTTACCTAACAAATCAAATGGGTTGGA-3′) containing N-terminal G4p2 coding sequence were annealed and inserted into BamHI-cleaved pGEX-3X. A suitable clone containing the properly oriented insert was cleaved with AatII (at the site in the oligomer sequence) and EcoRI, and a 2.1-kilobase DNA fragment derived from pUC19G4p2 cleaved with EcoRI and partially with AatII was inserted. The desired clone was identified by restriction site analysis and by sequencing. This clone should direct the synthesis of a protein identical to G4p2 in an amino acid sequence after removal of the glutathione S-transferase domain, except the initiator methionine is replaced by glycine-isoleucine. A large scale preparation of recombinant protein was prepared in the Escherichia coli host strain LE392 according to recommended procedure (24Smith D.B. Corcoran L.M. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Vol. 2. John Wiley & Sons, New York1992: 16.7.1-16.7.8Google Scholar). A yield of 0.75 mg of recombinant G4p2/liter of culture was obtained after proteolytic removal of the glutathione S-transferase domain with factor Xa.Data Base Searches and Sequence and Image Analysis ProgramsNon-redundant data base searches were carried out at the National Center for Biotechnology Information with the BLAST network service. Sequence manipulation and analysis was done with the GCG Sequence Analysis software package 7.2. Secondary structure prediction was done with PHD 1.0 (25Rost B. Sander C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7558-7562Google Scholar) via E-mail server. Silver-stained gels and autoradiographs were photographed and scanned onto PhotoCD (Kodak). Images were converted to PICT files with Photoshop 2.5.1 (Adobe), labeled with Canvas 2.1 (Deneba), and printed with a Phaser II SDX dye sublimation printer (Tektronix).RESULTSIdentification of a G4 Nucleic Acid Binding Protein, G4p2, in YeastWe have analyzed a yeast whole cell extract for binding activities specific for G4 DNA. Fig. 2 A shows a mobility shift assay with a parallel G4 DNA oligomer probe (Fig. 1 lists the oligonucleotides used) and excess double-stranded carrier DNA, demonstrating two major (G4p1 and G4p2) and several minor components of yeast that bind to GL(G4). (This paper deals with the characterization of G4p2; the characterization of G4p1 will be described elsewhere. 2J. D. Frantz and W. Gilbert, manuscript in preparation. 2) Protease and nuclease treatment of the extract showed that G4p2 is a protein without a nucleic acid component (data not shown). Fig. 2 B shows competition binding experiments with specific DNAs in single-stranded or parallel G4 form. Single-stranded DNAs do not compete, but DNAs containing parallel G4 regions surrounded by either As or Ts compete effectively. This demonstrates that the G4 domain of the probe is required and sufficient for stable binding to G4p2. Mobility shift experiments with extracts from several yeast strains showed that G4p2 is not restricted to strain SK1 (data not shown). We undertook the purification of G4p2 in order to characterize it further.Figure 2:Identification of G4 nucleic acid-specific binding proteins in a yeast extract. A, 5-fold serial dilutions of yeast extract were analyzed by mobility shift assay with 320 pg of probe as described under “Experimental Procedures.” Extract dilutions are indicated above the lanes. 1 μl of undiluted extract (25 μg) was analyzed in the left-hand lane. The G4 DNA probe and the two major G4 DNA binding proteins are indicated. B, the binding specificity of G4p2 was analyzed by mobility shift competition assays with 320 pg of probe and with 10-fold serial dilutions of unlabeled G4 and single-stranded competitor DNAs (see Fig. 1) as described under “Experimental Procedures.” 1 μl of a 20-fold dilution of extract (1.25 μg) was analyzed. Relative mass of competitor over probe is indicated above the lanes. GL(SS) indicates single-stranded GL oligomer, and sal DNA(SS) indicates single-stranded salmon sperm DNA.View Large Image Figure ViewerDownload (PPT)Purification of G4p2Small-scale batch experiments revealed that G4p2 binds to S-Sepharose at pH 11 in a sodium bicarbonate buffer and not in a sodium phosphate buffer. At pH 11, relatively few proteins in the yeast extract bind S-Sepharose, and fewer still are eluted by the buffer exchange. We used a G4 DNA affinity column as a second step. Table I outlines the purification and yields of G4p2. A decline in G4 DNA binding activity can be partially reversed by treating G4p2-containing fractions with 100 m M NaOH for several minutes just prior to loading G4p2-containing fractions on the G4 DNA affinity column. The final preparation contains 9.4 μg of G4p2/ml. In a subsequent purification of G4p2 (prep 2), we processed twice the amount of extract, and the concentration and buffer exchange step (Amicon) was omitted in favor of dialysis with improved yield (42 μg of G4p2/ml). Fig. 3 shows a SDS-PAGE gel of G4p2 containing fractions from the G4 DNA affinity column (prep 1), identifying a ∼32-kDa protein as G4p2. Quantitative estimates of G4p2 by mobility shift assay are in agreement with quantitative estimates of the ∼32-kDa protein by SDS-PAGE. The protein is essentially purified in fraction 22. G4p2 comprises 0.017% of total yeast protein by mass or about 35,000 molecules/cell and is therefore a moderately abundant protein.Table I:Purification of G4p2 Open table in a new tab Characterization of Purified G4p2Fig. 4 shows a quantitation of a series of competition experiments with the purified protein. G4p2 possesses equivalent affinities for parallel G4 DNA and RNA (compare GL(G4) with rGL(G4)). This establishes the possibility that, in vivo, this protein binds DNA and/or RNA. G4p2 has the highest affinity for molecules containing multiple G4 domains, with a 100-fold preference for parallel G4 DNA bearing two G4 domains over a similar molecule with just one domain (Fig. 4, compare TGT2(G4 ) with TGT(G4 )). Further, G4p2 displays a mild (3-fold) preference for antiparallel G4 DNA over parallel G4 DNA (Fig. 4, compare TGT2(G′2 ) with TGT(G4 )) and virtually no affinity for single-stranded and double-stranded DNA competitor (Fig. 4, compare GL(SS) and GL(DS) with GL(G4 )). The small amount of competition observed with high concentrations of single-stranded competitor (Fig. 4, GL(SS)) can be attributed to conversion of a fraction of the competitor into G4 structures during the course of the experiment.Figure 4:Binding specificity of purified G4p2 for G4 nucleic acids. Purified G4p2 was analyzed by mobility shift competition assays with 2 ng of probe and with serial dilutions of unlabeled competitor nucleic acids (see Fig. 1) as described under “Experimental Procedures.” Bound probe was measured by exposing dried gels to an imaging plate (Fuji). 1 μl of a 200-fold dilution of purifed G4p2 (210 pg, prep 2) was analyzed. Competitor nucleic acids are indicated. GL(SS) indicates a single-stranded GL oligomer, and GL(DS) indicates GL oligomer annealed to a complementary sequence.View Large Image Figure ViewerDownload (PPT)Incubations of purified G4p2 with parallel G4 DNA in the presence of various divalent cations and ATP indicate this protein, on its own, does not possess other activities, such as cleaving or unwinding activity, toward this substrate (data not shown). Fig. 5 shows that the equilibrium constant for G4p2 binding to parallel G4 DNA (GL(G4) is 2.2 × 108M−1in 50 m M KCl at pH 7.3 at room temperature. This value is comparable with those for other protein-DNA interactions, which involve proteins of moderate abundance (21Fried M.G. Crothers D.M. J. Mol. Biol. 1984; 172: 241-262Google Scholar).Figure 5:Equilibrium constant for G4p2 binding to GL(G4) . Mobility shift assays with 2.7 fm of probe and with 4-fold serial dilutions of G4p2 beginning with 1.35 pm were carried out as described under “Experimental Procedures.” Free probe was measured by exposing the dried gel to an imaging plate (Fuji). Total, bound, and free G4p2 was quantitated as described under “Experimental Procedures.” Free G4p2 concentration at 50% probe occupancy is indicated and is equivalent to l/Keq.View Large Image Figure ViewerDownload (PPT)Cloning and Sequencing of the G4p2 GeneIn order to obtain the primary sequence of G4p2, we determined to isolate its gene. We had sufficient material at hand to obtain a partial amino acid sequence of G4p2. An aliquot of pooled fractions 21-27 obtained from the S-Sepharose column was fractionated by SDS-PAGE and electrotransferred to nitrocellulose. G4p2 was excised, and the sequences of two internal peptides were obtained. A hybridization probe based on a reverse translated peptide sequence was used to screen a yeast genomic library in λEMBL3a, and three strongly hybridizing phage were isolated. A degenerate oligomer based on a reverse translated peptide sequence was used to obtain access into the G4p2 gene, and primer walking from the access point provided the remaining DNA sequence.Analysis of the G4p2 Peptide SequenceFig. 6 shows the DNA sequence encoding G4p2. It contains an open reading frame potentially encoding a protein of 273 amino acids with a calculated molecular mass of 29.9 kDa. The amino acid composition predicts a hydrophilic protein with an unusually high pI of 10.41, due in part to an abundance of lysine (11.4%). Five regions are characterized by skewed amino acid compositions. A region rich in proline (Fig. 6 A, (A)) is located at residues 38-54, and a region rich in serine and threonine (Fig. 6 A, (B)) is located at residues 77-102. Residues 119-141 are rich in alanine (Fig. 6 A, (C)), and residues 147-164 are rich in glutamine and asparagine (Fig. 6 A, (D)). Finally, a region containing mostly asparagine, arginine, and glycine (Fig. 6 A, (E)) is located at residues 225-245. Regions rich in glutamine or asparagine, such as regions (D) and (E) (Fig. 6 A), have been implicated in protein-protein contacts (26Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Google Scholar), whereas some arginine- and glycine-rich sequences, such as that in region (E) (Fig. 6 A), have been shown to bind RNA (27Kiledjian M. Dreyfuss G. EMBO J. 1992; 11: 2655-2664Google Scholar). The PHD secondary structure prediction algorithm (25Rost B. Sander C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7558-7562Google Scholar) predicts with an expected accuracy of 61% that G4p2 contains α-helices at residues 114-140 (α-helix I) and 147-159 (α-helix II). Helix I harbors the alanine-rich region (Fig. 6 A, (C)) described above. A segment of helix I containing residues 125-138 is strongly amphipathic, with one side of the helix carrying five acidic residues. Helix II, which contains the glutamine- and asparagine-rich region (Fig. 6 A, (D)), has an interesting “zipper” of alternating hydrophobic and glutamine or asparagine (amido) residues.Figure 6:Nucleotide sequence of the G4P2 gene and its deduced amino acid sequence. A, the underlined regions are segments of the protein that demonstrate skewed amino acid composition as described in the text. α-Helices I and II, identified by secondary structure prediction programs, are enclosed in curved brackets. B, a partial restriction map of the G4p2 gene.View Large Image Figure ViewerDownload (PPT)Results of Data Base SearchesA TBLASTN search of the non-redundant nucleotide sequence data base at the National Center for Biotechnology Information located two entries, MPT4 and STO1, potentially encoding gene products identical to G4p2. MPT4 (accession number D26183) was isolated with a screen designed to identify multicopy suppressors of staurosporine sensitivity associated with the POP2 mutation in yeast.3 3A. Sakai, personal communication. The POP2 mutation itself was originally identified in a screen for genes required for the repression of gene expression in the absence of glucose (6Sakai A. Chibazakura T. Shimizu Y. Hishinuma F. Nucleic Acids Res. 1992; 20: 6227-6233Google Scholar). STO1 (accession number D32208) was isolated as a multicopy suppressor of TOM1, a molecularly uncharacterized mutation in yeast that leads to temperature-sensitive cell cycle arrest at the G2/M boundary.4 4Y. Kikuchi, personal communication. No other polypeptide sequences with significant homologies to G4p2 have been identified thus far. Searches of the PROSITE 8.0 data base failed to identify peptide motifs of biological interest in the G4p2 amino acid sequence.Analysis of Recombinant G4p2As proof that we had identified the correct protein and gene, we prepared a recombinant version of G4p2. Bacterially synthesized G4p2 was produced as a fusion to glutathione S-transferase. The protein was" @default.
• W2018172829 created "2016-06-24" @default.
• W2018172829 creator A5051655733 @default.
• W2018172829 creator A5080862743 @default.
• W2018172829 date "1995-04-01" @default.
• W2018172829 modified "2023-10-03" @default.
• W2018172829 title "A Yeast Gene Product, G4p2, with a Specific Affinity for Quadruplex Nucleic Acids" @default.
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