©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Yeast Gene Product, G4p1, with a Specific Affinity for Quadruplex Nucleic Acids (*)

(Received for publication, April 28, 1995)

J. Daniel Frantz (§) Walter Gilbert

From the Department of Molecular and Cellular Biology, Harvard University, Biological Laboratories, Cambridge, Massachusetts 02138

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

G4 nucleic acids are four-stranded helical structures that are formed in vitro by nucleic acids that contain guanine tracts. These structures anneal readily under physiological conditions and are unusually stable once formed. G4 nucleic acids are thought to participate in telomere function, retroviral genome dimerization, chromosome alignment during homologue pairing, and mitotic recombination, although the in vivo demonstration of these structures in any of these situations has not yet been achieved. Here we purify and characterize an activity from yeast, G4p1, which has a high and specific affinity for G4 nucleic acids. G4p1 prefers substrates containing multiple G4 domains, has an equal affinity for parallel and antiparallel G4 structures, and binds equivalently to RNA and DNA in G4 form. The Keq for G4p1 binding to a G4 DNA oligomer is 5.0 10^8M, under near physiological conditions. G4p1 was purified and shown to derive from a 42-kDa protein (p42). We have cloned and sequenced the gene encoding p42 and show it to encode a novel protein with a region significantly homologous to bacterial methionyl-tRNA synthetase dimerization domains. We have reconstituted the G4p1 binding activity with recombinant p42 and present evidence that G4p1 is a homodimer of p42.


INTRODUCTION

Nucleic acids containing guanine tracts will associate in vitro into four-stranded right-handed helices, stabilized by guanine base quartets (for review see (1, 2, 3, 4, 5) ). These helices exist in parallel and cis or trans antiparallel conformations, as revealed by NMR and x-ray analysis. These quadruplex structures are called the G4 nucleic acids. G4 structures, once formed, are exceptionally stable, particularly in the presence of certain alkali metal cations, such as potassium, which efficiently bind guanine carbonyl oxygens lining the axial cavity of the helix. G4 helices can form at appreciable rates, particularly into the monomeric or dimeric antiparallel conformers, under moderate physiological conditions.

Do G4 structures arise in vivo? This is presently an unanswered question. The simple sequence organization of telomeres, specifically the guanine tracts, are strongly conserved phylogenetically(6) , indicating that telomere function depends in some way on a property unique to these tracts. Oligomers containing telomere repeat sequences readily form G4 structures in vitro(7, 8, 9) , and, a G4 DNA annealing activity, which greatly accelerates the formation of G4 structures by these oligomers, was identified in an Oxytricha telomere binding protein(10) . There is similar in vitro evidence supporting a role for G4 structures in the dimerization of retroviral genomes during virion assembly(11, 12) .

Sep1/Kem1 is a complex yeast enzyme which, among other activities, possesses a nucleolytic activity specific for DNA oligomers containing G4 domains(13) . Null alleles of the gene encoding this enzyme lead, in part, to mitotic loss of chromosomes and meiotic arrest at pachytene (14) . These defects may be due to an inability of the mutant cell to process properly G4 structures that may arise during the course of chromosomal replication, recombination, or meiotic pairing. Also, a domain in the 3`-untranslated region of the insulin-like growth factor II mRNA, has been shown in vitro to exist in a G4 structure(15) . This transcript undergoes a specific cleavage reaction in vivo at a site just 5` to this domain, suggesting a role for G4 structures in targeting mRNA-processing events.

To explore the possible biological functions of G4 nucleic acids, we analyzed a yeast extract for activities specific to these structures. We have identified an activity, G4p1, with a high and specific affinity for G4 nucleic acids. The activity was purified and shown to derive from a 42-kDa protein (p42). The gene encoding p42 was isolated and sequenced and shown to encode a novel protein with a domain significantly homologous to bacterial methionyl-tRNA synthetase dimerization domains. G4p1 is probably a homodimer of p42.


MATERIALS AND METHODS

Preparation of G4 Oligomers

DNA and RNA oligomers (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(16) , and stored in 10 mM Tris, pH 7.6, 1 mM EDTA, 100 mM KCl at 4 °C. Yields were determined by absorbance at 260 nm.


Figure 1: G4 oligomers used in mobility shift and competition experiments. Guanines in boldface are those that participate in quartet formation. G(4) indicates tetrameric parallel (p) structures, and G`(2) indicates dimeric antiparallel (ap) structures (nomenclature according to Sen and Gilbert(2) ).



Mobility Shift Assays, Competitions, and SDS-PAGE^1

These procedures have been described previously(17) . Essentially, 5000 counts/s of 5`-labeled GL(G(4)) (320 pg, unless otherwise indicated) was added to extract or G4p1 fractions in binding buffer (20 mM NaHepes, pH 7.3, 1 mM EDTA, 50 mM KCl, 5 mM NaPO(4), 0.05% Triton X-100, 5 mM BME, 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. After 10 min at room temperature, the reactions were loaded on 5% polyacrylamide gels and run in 50 mM TBE (50 mM Tris borate, 1 mM EDTA, pH 8.2), 1 mM KCl at 12.5 V/cm. The gels were dried and exposed to x-ray film (Kodak), or to an imaging plate (Fuji).

Competition experiments were carried out as above, with saturating amounts of GL(G(4)) probe. Ten-fold serial dilutions of competitor nucleic acids were premixed with extract or G4p1 fractions in binding buffer just prior to adding the probe. Relative mass amounts of competitor to probe ranged from 0.1 to 1000.

SDS-PAGE was carried out by standard procedures(18) . Ten-µl aliquots of G4p1 fractions were loaded on 10% SDS-PAGE gels, and protein bands were visualized by staining twice with silver (Bio-Rad).

Large Scale Preparation of Yeast Extract

The preparation of this extract has been described previously(17) . Essentially, a mid-log culture (50 liters) of yeast (Saccharomyces cerevisiae, strain SK1) was harvested and lysed in liquid nitrogen according to published procedures(19) . Proteins were extracted from lysed cells in extraction buffer (20 mM NaHepes, pH 7.3, 1 mM EDTA, 10 mM MgCl(2), 420 mM KCl, 0.05% Triton X-100, 5 mM BME, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A). The extract was cleared and dialyzed against buffer A (20 mM NaHepes, pH 7.3, 1 mM EDTA, 45 mM KCl, 0.05% Triton X-100, 5 mM BME, 10% glycerol). The dialysate was cleared again 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.

Purification of G4p1

All extract and G4p1 fraction manipulations were done at 4 °C. Yeast extract (1.25 g in 50 ml) was thawed and cleared by centrifugation at 10,000 g for 10 min. The supernatant (900 mg) was loaded at 25 ml/h on a 25-ml phosphocellulose (Whatman) 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 with a 140-ml linear gradient of increasing NaPO(4), pH 7.3, concentration (0-500 mM). One-ml fractions were collected and analyzed by mobility shift assay for G4p1 and by SDS-PAGE. Fractions 122-137 (16 ml), containing G4p1, were pooled and dialyzed (Spectra/Por 7; molecular weight cut-off, 3500) for 12 h against 1600 ml of buffer B (20 mM sodium citrate, pH 6.0, 1 mM EDTA, 45 mM KCl, 0.05% Triton X-100, 5 mM BME, 10% glycerol).

Partially purified G4p1 (16 ml) from the previous step was loaded at 25 ml/h on a 20-ml S Sepharose FF (Pharmacia Biotech Inc.) bed, which had been equilibrated in buffer B and packed in a 2.5 10-cm EconoColumn (Bio-Rad). The bed was washed with 100 ml of buffer B, and proteins were eluted with an 80-ml linear gradient of increasing NaCl concentration (0-600 mM). One-ml fractions were collected and analyzed by mobility shift assay for G4p1 and by SDS-PAGE. Fractions 48-61 (12 ml), containing G4p1, were pooled and dialyzed for 12 h against 1200 ml of buffer C (100 mM BisTris-Cl, pH 6.0, 1 mM EDTA, 0.05% Triton X-100, 5 mM BME, 10% glycerol).

Partially purified G4p1 (12 ml) from the previous step was loaded at 25 ml/h on a DEAE-Sepharose FF (Pharmacia) bed, which had been equilibrated in buffer C and packed in a 2.5 10-cm EconoColumn (Bio-Rad). The bed was washed with 100 ml of buffer C, and proteins were eluted with simultaneous 60-ml linear gradients of decreasing BisTris-Cl, pH 6.0, concentration (100-0 mM) and increasing NaPO(4), pH 7.3, concentration (0-200 mM). One-ml fractions were collected and analyzed by mobility shift assay for G4p1 and by SDS-PAGE. Fractions 38-49 (12 ml), containing G4p1, were pooled and dialyzed for 12 h against 1200 ml of buffer D (20 mM NaHepes, pH 7.3, 1 mM EDTA, 50 mM KCl, 0.05% Triton X-100, 5 mM BME, 10% glycerol).

A 1-ml G4 DNA affinity bed was prepared as described previously(17) . Essentially, oligomer GL (Fig. 1), with biotin incorporated next to the 3` end, was synthesized at 1 µmol scale and converted into G4 form. The G4 DNA (3.95 mg) was bound to 2 ml of 50% streptavidin-agarose (Sigma) in 10 mM Tris, pH 7.6, 1 mM EDTA, 100 mM KCl. The affinity matrix was washed twice in buffer D, 100 mM NaCl, packed in a 0.7 5 cm EconoColumn (Bio-Rad), and equilibrated at 4 °C.

Partially purified G4p1 (12 ml) from the previous step was loaded at 2.3 ml/h on the G4 DNA affinity column, washed with 5 ml of buffer D, 100 mM NaCl, and bound proteins were eluted with a 6-ml convex exponential gradient of increasing NaCl concentration (100-500 mM). Sixty 0.1-ml fractions were collected and assayed by mobility shift assay for G4p1 and by SDS-PAGE. Fractions 33-42 (1 ml), containing G4p1, were pooled.

Determination of the Keq for G4p1 Binding to G4 DNA

Mobility shift experiments as described above were carried out with 1.25 fm of GL(G(4)) probe (Fig. 1), and with 2-fold serial dilutions of G4p1 (40, 20, 10, and 5 fm). A second preparation (prep 2) of G4p1 was used here, prepared as described except that the DEAE column was omitted, resulting in a 10-fold reduction in purity as judged by SDS-PAGE. Stock G4p1 was quantitated by titrating a dilution of G4p1 with increasing amounts of precisely quantitated GL(G(4)) DNA probe, until binding to G4p1 was saturated. At saturation, the molarity of the bound probe is equivalent to that of G4p1. By this method, prep 2 contains 40 nM G4p1. The dried gels were exposed to an imaging plate (Fuji), and the amount of free probe at each G4p1 dilution was measured. Keq was estimated from a plot of log (free G4p1 concentration) versus log (bound probe (= total probe - free probe)/free probe)(20) . The free G4p1 concentration was estimated by subtracting the concentration of bound probe at each dilution, which equals the concentration of bound G4p1, from that of input G4p1.

Peptide Sequencing and DNA Hybridization Probes

Purified G4p1 (0.4 ml), obtained from the G4 DNA affinity 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. The 42-kDa polypeptide was excised and sequenced (Harvard Microchemistry Facility). The sequences of two internal tryptic peptides (NT-57, GDYMQNLLEVSSTDKLEINHDL; NT-59, DNTFIVSTLYPTSTDVHVFEVAL) were obtained. A unique oligomer, incorporating most frequent codon usage information for yeast(21) , was synthesized based on reverse-translated NT-59 (5`-TTCATTGTTTCTACTTTGTACCCAACTTCTACTGATGTTCATGTTTTCGAAGTTGC-3`). This hybridization probe was tested on Southern blots of yeast genomic DNA and found suitable for library screening.

Isolation, Subcloning, and Sequencing of the Gene Encoding p42

Oligomer NT-59, labeled with [-P]ATP and T4 polynucleotide kinase, was used to screen 6 10^3 plaques of EMBL3a containing a yeast genomic library(22) . Three positive plaques were further purified. These were mapped by agarose gel electrophoresis and transfer to nylon membranes of partial restriction enzyme digests of SmaI-cleaved phage DNA (the SmaI sites are located in each arm of the vector, near the insert), followed by hybridizations with mapping oligomers that anneal to phage DNA flanking the SmaI sites. The location of the p42 coding sequence within the inserts was determined by hybridization to these membranes with oligomer NT-59. A 6-kb EcoRI fragment containing the p42 coding sequence was subcloned into pBluescript-SK+ (Stratagene), producing pSK42. The nucleotide sequence of the p42 coding region was determined by a combination of ExoIII/mung bean nuclease nested deletion and primer walking strategies.

Preparation of Recombinant p42 and p85

Bacterially expressed p42 and p85 fusions to glutathione S-transferase were produced with the pGEX-5X-1 (Pharmacia) vector system. A 2.0-kb ApaI/BamHI fragment from pSK42 was subcloned into ApaI/BamHI-cleaved pBluescript-KS+ (Stratagene). This construct was partially cleaved with Csp45I (NspV), and the ends were made blunt by treatment with T4 DNA polymerase in the presence of deoxyribonucleotides. Subsequent cleavage with NotI yielded a 1.9-kb fragment, which was inserted into SmaI/NotI-cleaved pGEX-5X-1. The polypeptide produced by this construct should be identical in amino acid sequence to p42 after removal of the glutathione S-transferase domain, except the N-terminal residues MSDLVTKF are replaced by GIPEFP.

A 3.5-kb Sal partial/SmaI fragment from pSK85 (^2)was subcloned into SmaI/SalI-cleaved pBluescript-KS+ (Stratagene). This construct was cleaved with AseI, and the ends were made blunt by treatment with T4 DNA polymerase in the presence of deoxyribonucleotides. Subsequent cleavage with NotI yielded a 2.5-kb fragment, which was inserted into SmaI/NotI cleaved pGEX-5X-1. The polypeptide produced by this construct should be identical in amino acid sequence to p85 after removal of the glutathione S-transferase domain, except that the N-terminal residues MTKLFSKVKESIEGIKMPSTLTI are replaced by GIPEFP.

Large scale preparations of recombinant proteins were prepared in the Escherichia coli host strain LE392, according to recommended procedure(24) . Yields of 35 µg of GSTp42 and 75 µg of GSTp85 per 100 ml of culture were obtained. The glutathione S-transferase domains were removed from p42 and p85 by proteolysis with factor Xa, according to recommended procedure(24) .

Data Base Searches, Sequence, and Image Analysis Programs

Nonredundant data base searches were carried out at the National Center for Biotechnology Information (NCBI) with the BLAST network service. Sequence manipulation and analysis were done with the Genetics Computer Group, Inc. (GCG) Sequence Analysis Software Package 7.2. Stained gels and autoradiographs were photographed and scanned onto PhotoCD (Kodak). Images were converted into PICT files with Photoshop 2.5.1 (Adobe) and labeled with Canvas 2.1 (Deneba), and prints were prepared with a Phaser II SDX dye sublimation printer (Tektronix).


RESULTS

Identification of a G4 Nucleic Acid Binding Protein, G4p1, in Yeast-We have tested extracts for activities specific for G4 quadruplex DNA. Previously, we reported on the characterization of G4p2, a protein present in a yeast whole-cell extract that binds specifically to nucleic acids containing G4 domains(17) . Here we describe another such binding activity, G4p1, found in the same extract preparation. Fig. 2A shows a mobility shift experiment identifying both the G4p1 and G4p2 binding activities. G4p1 binds to a parallel G4 DNA probe in the presence of excess double-stranded DNA ( Fig. 1lists the oligonucleotides used). Fig. 2B shows mobility shift competition experiments with single-stranded competitors and with competitors containing G4 structures. Single-stranded GL and denatured salmon sperm DNA do not compete, but molecules containing G4 regions compete effectively, identifying the G4 domain of the probe as required and sufficient for stable binding to G4p1. Mobility shift experiments with extracts from other yeast strains determined that G4p1 is not restricted to SK1 (data not shown). We proceeded to purify G4p1 in order to characterize it more fully.


Figure 2: Identification of G4 nucleic acid specific binding activities in a yeast extract. A, 5-fold serial dilutions of yeast extract were analyzed by mobility shift assay with 320-pg probe as described. Extract dilutions are indicated above the lanes. One µl of undiluted extract (25 µg), was analyzed in the leftmost lane. The G4 DNA probe and the two major G4 DNA binding activities, G4p1 and G4p2, are indicated. B, the binding specificity of G4p1 was analyzed by mobility shift competition assays, with 320-pg probe and with 10-fold serial dilutions of unlabeled G4 and single-stranded competitor DNAs (see Fig. 1) as described. One 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.



Purification of G4p1

We determined a purification strategy for G4p1 with small scale batch experiments, where binding matrix, buffer ion, and pH were varied to identify conditions for the binding and elution of the activity. Table 1outlines the four-step purification and yields. The final fraction contains approximately 13 µg of protein in 1 ml. Fig. 3shows an SDS-PAGE gel of fractions obtained from the G4 DNA affinity column, identifying two proteins, with apparent molecular weights of 85 kDa (p85) and 42 kDa (p42), eluting with the G4p1 activity. Further fractionation by gel filtration showed that p42, p85, and the G4p1 activity co-elute with apparent molecular weights of 200-240 kDa, suggesting that p42 and p85 may be complexed (data not shown). This possibility is further discussed in the following section. As described below, we were able to fully reconstitute the G4p1 activity in vitro with recombinant p42. Therefore, we present here the characterization of p42, which contains the G4p1 activity, and describe p85 elsewhere.^2 The final fraction contains 4.6 µg of p42 as determined by quantitative amino acid analysis of p42 excised from an SDS-PAGE gel. p42 comprises 0.002% of total yeast protein by mass, or about 3000 molecules/cell.




Figure 3: Purification of G4p1. A, fractions from the G4 affinity column were analyzed by mobility shift assay with 64 pg probe, as described. One µl of a 20-fold dilution of each fraction was analyzed. The input fraction (IN) is comprised of pooled and concentrated DEAE-Sepharose fractions 38-49. G4p1 is indicated. B, SDS-PAGE of the same fractions as in A. In each case, 10 µl of undiluted fraction was analyzed. p42, p85, and the molecular weights of markers are indicated. The gel was stained twice with silver.



Characterization of Purified G4p1

Fig. 4shows the results of various competition experiments with purified G4p1. The activity demonstrates equivalent affinities for parallel G4 RNA and DNA (compare rGL(G(4)) with GL(G(4))), establishing the possibility that G4p1 is either a DNA and/or RNA binding protein in vivo. G4p1 demonstrates highest affinities for substrates with at least two G4 domains. G4p1 has a 60-fold preference for molecules containing two parallel G4 domains over substrates with one domain (compare TGT2(G(4)) with TGT(G(4))), Also, G4p1 demonstrates equivalent affinity for antiparallel and parallel G4 structures (compare TGT2(G`(2)) with TGT(G(4))). Virtually no affinity for double-stranded or single-stranded competitor is displayed (compare GL(SS) and GL(DS) with GL(G(4))). Competition observed at high concentrations of single-stranded competitor (GL(SS)) can be attributed to conversion of a fraction of the competitor into G4 structures during the course of the experiment. Incubations of purified G4p1 with G4 DNA substrates in the presence of various divalent ions and ATP do not disclose any additional activities, such as cleaving or unwinding, toward these substrates (data not shown).


Figure 4: Binding specificity of purified G4p1 for G4 nucleic acids. Purified G4p1 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. Bound probe was measured by exposing dried gels to an imaging plate (Fuji). One µl of a 20-fold dilution of purified G4p1 (500 pg; prep 2) was analyzed. Competitor nucleic acids are indicated. GL(DS) indicates GL oligomer annealed to a complementary sequence.



Fig. 5shows that the equilibrium constant for G4p1 binding to parallel G4 DNA (GL(G(4))) is 5.0 10^8M, in 50 mM KCl at pH 7.3 and at room temperature. This value is comparable with those for other known protein-DNA interactions.


Figure 5: Equilibrium constant for G4p1 binding to GL(G(4)). Mobility shift assays with 1.25-fm probe and with 2-fold serial dilutions of G4p1, beginning with 40 fm, were carried out, as described. Free probe was measured by exposing the dried gel to an imaging plate (Fuji). Total, bound, and free G4p1 was quantitated as described under ``Materials and Methods.'' Free G4p1 concentration at 50% probe occupancy is indicated and is equivalent to 1/Keq.



Cloning and Sequencing of the Gene Encoding p42

In order to obtain the amino acid sequence of p42, we isolated and sequenced its gene. We had sufficient material at hand to obtain a partial amino acid sequence of p42. An aliquot of pooled fractions 33-42 obtained from the G4 DNA affinity column was fractionated by SDS-PAGE and electrotransferred to nitrocellulose. The p42 was excised, and the sequences of two internal peptides were obtained. A hybridization probe based on reverse-translated peptide sequence was used to screen a yeast genomic library in EMBL3a, and three strongly hybridizing phage were isolated. The p42 coding region in each phage was mapped, one such region was subcloned into the pBluescript-SK+ vector, and its nucleotide sequence was determined.

Analysis of the p42 Peptide Sequence

Fig. 6shows the nucleotide sequence encoding p42, identifying an open reading frame that potentially encodes a protein of 375 amino acids, with a calculated molecular weight of 42 kDa. Both peptide sequences obtained by internal peptide sequencing are represented, indicating the gene was correctly identified. Extensive homology searches of the available nucleotide and peptide sequence data bases established that p42 is a novel protein. Three very divergent data base entries were identified, however, with partial matches to a common region of p42.


Figure 6: Sequence analysis of p42. A, nucleotide sequence of the gene encoding p42 and its deduced amino acid sequence. alpha-helixes I and II, identified by GCG's Robson-Garnier secondary structure prediction programs, and a putative dimerization domain, are enclosed in brackets. B, a partial restriction map of the gene encoding p42.



Significant homology was detected between the C-terminal domains of bacterial methionyl-tRNA synthetases and an internal region of p42. Sequence alignments with GCG's GAP program show that a 36% identity exists between residues 522-616 of the Thermus aquaticus methionyl-tRNA synthetase (SwissProt accession number P23395) and residues 208-304 of p42. Similar matches of comparable quality were found to the Bacillus stearothermophilus (SwissProt accession number P23920) and E. coli (SwissProt accession number P00959) enzymes. These matches lie outside the catalytic core of the methionyl-tRNA synthetases, in a region identified as the dimerization domain of these enzymes, which exist, in bacteria, as homodimers(25) . This region of p42, then, may be a dimerization domain.

The C-terminal segment of human endothelial monocyte-activating polypeptide (EMAP) II (GenBank accession number U10117), residues 152-311, are 50% identical to residues 205-364 of p42. The specific function of this region of EMAP II is unknown; however, by inferring from the matches noted above, EMAP II may also dimerize. EMAP II is secreted by certain human and mouse cultured tumor cells and can induce an acute host inflammatory response(23) . The N-terminal segments of these proteins are responsible for their inflammation-inducing properties.

An expressed sequence tag derived from rice callus (DDBJ accession number D23020), encodes a protein fragment 120 amino acids in length with a 61% identity to residues 185-302 of p42. The remainder of the cDNA sequence is required to establish whether it encodes a homologue of p42.

An inspection of the amino acid sequence of p42 reveals two potentially interesting structural elements. Residues 127-160 (I) and 166-199 (II) are predicted to exist as alpha-helices by GCG's Robson-Garnier secondary structure prediction program. Helix I is lysine rich at both ends, and helix II is N-terminally rich in charged residues and alanine and C-terminally rich in glutamine. Neither helix is strongly amphipathic.

Analysis of Recombinant G4p1

Initially, it was unclear which of p42 and/or p85 was responsible for the G4p1 activity. To resolve this issue, we expressed p42 and p85 separately as fusions to glutathione S-transferase in bacteria. The proteins were purified from extracts with a glutathione-agarose matrix and eluted. SDS-PAGE of the purified fusion proteins demonstrates homogeneous bands with the expected mobilities (data not shown). Fig. 7shows a mobility shift assay demonstrating that GSTp42 can bind G4 DNA, whereas GSTp85 cannot. Once released from the glutathione S-transferase domain by treatment with factor Xa, p42 displays two binding activities of differing mobility. These are most likely monomers and dimers of p42. Since the activity with the lower mobility migrates at the same rate as G4p1, G4p1 is most likely a dimer of p42. The p42 monomer, indicated by the activity with the higher mobility, retains the G4 DNA binding property. Addition of p85 to p42 does not modulate the binding activity displayed by p42, showing that, under these conditions, these proteins do not interact. Purified G4p1, then, is a stable dimer of p42, since we do not see evidence in the yeast extract of the binding activity indicative of a p42 monomer.


Figure 7: Reconstitution of G4p1 with recombinant p42. Mobility shift assays were carried out as described, with GSTp42 and GSTp85 fusion proteins purified from E. coli. Lane1 contains no protein. Lanes2 and 5 contain 1 µl (70 ng) of GSTp42, and GSTp42 treated partially with factor Xa, respectively. Lanes4 and 7 contain 1 µl (150 ng) of GSTp85, and GSTp85 treated partially with factor Xa, respectively. Lanes3 and 6 contain mixtures (0.5 µl each) of GSTp42 and GSTp85, and GSTp42 and GSTp85 treated partially with factor Xa, respectively. Lane8 contains 1 µl of a 20-fold dilution of pooled fractions, containing G4p1, obtained from the G4 affinity column. The binding activities assigned to GSTp42, dimers of p42, and monomers of p42 are indicated.




DISCUSSION

G4p1, isolated from yeast, displays a selective affinity for nucleic acids in the G4 quadruplex form. We demonstrate this with competition mobility shift experiments at saturating levels of G4 probe and excess double-stranded DNA using specific nucleic acid competitors containing single-stranded, double-stranded, or G4 regions. G4p1 binds G4 domains in both RNA and DNA with equivalent affinity. G4p1 appears to be a dimeric protein with two identical 42-kDa (p42) subunits. p42 is a novel protein of unknown cellular function, with an internal region that shows significant homology to bacterial methionyl-tRNA synthetase dimerization domains.

Methionyl-tRNA synthetases of eubacteria contain a C-terminal domain that can be removed by proteolysis without affecting the catalytic properties of these enzymes(25) . Removal of this domain converts these usually homodimeric proteins into monomers, indicating that this domain is a module that mediates homodimerization. The discovery of domains homologous to this module in p42, a yeast protein, in a secreted mammalian cytokine, and in a protein from a higher plant, raises interesting questions concerning its evolutionary history.

As described above, a second protein, p85, purified along with p42 throughout the purification procedure. These proteins also co-fractionate on a gel filtration column with apparent molecular weights of 200-240 kDa, indicating they may be complexed. As described in this work, however, we have reconstituted the G4p1 activity with recombinant p42 alone. An interesting possibility is that p42 and p85 are in fact associated in a complex and dissociate upon binding of the G4 DNA probe by p42. This is presently under investigation. We have identified p85 as the yeast cytosolic glutamyl-tRNA synthetase.^2 If we can verify that p85 and p42 are complexed, this would implicate a G4 nucleic acid component in the translation process.

G4p1 has several features in common with another G4 nucleic acid binding activity, G4p2, which we have described previously(17) . Like G4p2, G4p1 binds parallel and antiparallel G4 nucleic acids, prefers substrates containing more than one G4 domain, and binds RNA as well and DNA in G4 form. Also, the affinities displayed by these activities for these substrates are similar. These common binding properties may indicate that these activities interact with similar substrates in vivo. We have speculated that G4p2 functions as a gene regulating factor, controlled by protein kinases, by interacting with regulatory DNA sequences, with mRNAs, or with structural RNAs involved in translation, via G4 structures. On the other hand, p42 shows little sequence similarity to the polypeptide responsible for the G4p2 activity, and there is no obvious common linear motif that might be responsible for their G4 nucleic acid binding properties. alpha-helix I and alpha-helix II, which lie just N-terminal to the putative dimerization module, may be the structural elements that bind G4 nucleic acids. Further studies on the biochemistry and genetics of these proteins are required to illuminate their cellular function.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM41895 (to W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
To whom correspondence should be addressed: Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215. Tel.: 617-732-2529; Fax: 617-732-2593.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; BME, betamercaptoethanol; kb, kilobase(s); GST, glutathione S-transferase.

(^2)
Information on the cloning, restriction enzyme mapping, and nucleotide sequence determination of the gene encoding p85 has been submitted for publication (J. D. Frantz and W. Gilbert).


ACKNOWLEDGEMENTS

We thank members of the Gilbert Lab for very useful discussion and Nancie Munroe and Lloyd Schoenbach for invaluable administrative and computer assistance. We also thank Michael Snyder for the yeast genomic library.


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