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G4 Resolvase 1 Binds Both DNA and RNA Tetramolecular Quadruplex with High Affinity and Is the Major Source of Tetramolecular Quadruplex G4-DNA and G4-RNA Resolving Activity in HeLa Cell Lysates

2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês

10.1074/jbc.m806277200

ISSN

1083-351X

Autores

Steven D. Creacy, Eric D. Routh, Fumiko Iwamoto, Yoshikuni Nagamine, Steven A. Akman, James P. Vaughn,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117–38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent Kd values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 ± 6 and 77 ± 6pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures. Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117–38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent Kd values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 ± 6 and 77 ± 6pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures. The nucleobase guanine is unique among bases in nucleic acids because of its great tendency toward self-association. Stable cyclic guanine quartets form by self-assembly through Hoogsteen hydrogen bonding between each of the four guanine molecules (N1-H and N2-H share hydrogen with O6 and N7 of the adjacent guanine). In the presence of physiological monovalent cations these quartets can self-associate into vertical stacks that coordinately bond a cation at the center of the G quartets, and the assemblies are further stabilized through stacking interactions (reviewed in Ref. 1.Davis J.T. Angew. Chem. Int. Ed. Engl. 2004; 43: 668-698Crossref PubMed Scopus (1447) Google Scholar). This vertical assembly of G quartets is known as G-quadruplex; this self-associating phenomenon is responsible for the observation that over 30 different derivatives of guanine can form gels in water (2.Guschlbauer W. Chantot J.F. Thiele D. J. Biomol. Struct. Dyn. 1990; 8: 491-511Crossref PubMed Scopus (413) Google Scholar). These are also the forces that stabilize the formation of quadruplex structures in nucleic acids that are also termed G4-DNA and G4-RNA. Since the initial observation by Sen and Gilbert (3.Sen D. Gilbert W. Nature. 1988; 334: 364-366Crossref PubMed Scopus (1538) Google Scholar) of the formation of tetramolecular G4-DNA, it has been shown that DNA or RNA molecules possessing runs of as few as three guanines in a row can form a variety of extraordinarily stable quadruplex structures (reviewed in Ref. 4.Arthanari H. Bolton P.H. Chem. Biol. 2001; 8: 221-230Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In fact, guanine quadruplex structures create the highest thermodynamically stable secondary structure per base pair participant found in nucleic acids. Formation of quadruplex structures in vivo could disrupt normal cellular metabolic processes such as DNA replication and recombination and RNA translation and processing; therefore, it might be expected that genome usage of runs of guanines would be minimized. However, guanine-rich regions capable of quadruplex structure formation are commonly found in the human genome. Two bioinformatic analyses using very different approaches to identify potentially stable intramolecular quadruplex forming sequences have each estimated that over 375,000 such sequences may exist in the human genome (5.Todd A.K. Johnston M. Neidle S. Nucleic Acids Res. 2005; 33: 2901-2907Crossref PubMed Scopus (810) Google Scholar, 6.Huppert J.L. Balasubramanian S. Nucleic Acids Res. 2005; 33: 2908-2916Crossref PubMed Scopus (1352) Google Scholar). Moreover, there is strong evidence that runs of guanines capable of forming quadruplex structures occur at genetic control regions, supporting the idea that quadruplex structures in DNA and RNA are a common part of the natural gene control repertoire. A bioinformatics database has registered 54,252 predicted G-quadruplexes near mRNA splicing and polyadenylation sites within human and mouse genes (7.Kostadinov R. Malhotra N. Viotti M. Shine R. D'Antonio L. Bagga P. Nucleic Acids Res. 2006; 34: D119-D124Crossref PubMed Scopus (71) Google Scholar). Recent human genome bioinformatics studies have found that promoter regions of the human genome are enriched in quadruplex forming sequences (8.Huppert J.L. Balasubramanian S. Nucleic Acids Res. 2007; 35: 406-413Crossref PubMed Scopus (1010) Google Scholar) as well as the first intron region of many genes, especially at a region about 200 bases downstream of the start of transcription (9.Eddy J. Maizels N. Nucleic Acids Res. 2008; 36: 1321-1333Crossref PubMed Scopus (222) Google Scholar). Interestingly, sequence analysis has also shown that within the human genome the potential of quadruplex formation is significantly higher in oncogenes and lower in tumor suppressor genes (10.Eddy J. Maizels N. Nucleic Acids Res. 2006; 34: 3887-3896Crossref PubMed Scopus (406) Google Scholar). In addition to these theoretical studies that highlight the prevalence of sequences with potential to form quadruplex structures, a number of empiric studies directly show long lived quadruplex structures in vivo. Some of this body of work includes: development of quadruplex specific antibodies that have detected quadruplex structures in the telomeres of the ciliate macronucleus of Stylonychia lemnae (11.Schaffitzel C. Berger I. Postberg J. Hanes J. Lipps H.J. Pluckthun A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8572-8577Crossref PubMed Scopus (524) Google Scholar, 12.Paeschke K. Juranek S. Simonsson T. Hempel A. Rhodes D. Lipps H.J. Nat. Struct. Mol. Biol. 2008; 15: 598-604Crossref PubMed Scopus (125) Google Scholar), the demonstration of a quadruplex binding-dependent fluorescence emission wavelength shift of a carbazole derivative in the telomeres of human chromosomes (13.Chang C.C. Chu J.F. Kao F.J. Chiu Y.C. Lou P.J. Chen H.C. Chang T.C. Anal. Chem. 2006; 78: 2810-2815Crossref PubMed Scopus (84) Google Scholar), and the construction of an inducible transcript in Escherichia coli incorporating the mammalian immunoglobin Sμ and Sγ3 switch regions that forms a cleavable quadruplex structure in the G-rich nontemplate strand of the gene upon induction of transcription (14.Duquette M.L. Handa P. Vincent J.A. Taylor A.F. Maizels N. Genes Dev. 2004; 18: 1618-1629Crossref PubMed Scopus (415) Google Scholar). More indirect empiric evidence for the existence of quadruplex structures in control regions is found in a number of specific genetic control elements that readily form quadruplex structures in vitro under physiological salt conditions including: the aforementioned telomeres (15.Parkinson G.N. Lee M.P. Neidle S. Nature. 2002; 417: 876-880Crossref PubMed Scopus (1737) Google Scholar, 16.Phan A.T. Luu K.N. Patel D.J. Nucleic Acids Res. 2006; 34: 5715-5719Crossref PubMed Scopus (275) Google Scholar), the immunoglobin heavy chain switch region (3.Sen D. Gilbert W. Nature. 1988; 334: 364-366Crossref PubMed Scopus (1538) Google Scholar), guanine-rich regions of ribosomal DNA (17.Hanakahi L.A. Sun H. Maizels N. J. Biol. Chem. 1999; 274: 15908-15912Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), the d(pCGG) repeats of the fragile-X mental retardation gene (18.Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), and the promoters of a number of proliferation associated genes such as c-MYC (19.Simonsson T. Pecinka P. Kubista M. Nucleic Acids Res. 1998; 26: 1167-1172Crossref PubMed Scopus (542) Google Scholar, 20.Siddiqui-Jain A. Grand C.L. Bearss D.J. Hurley L.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11593-11598Crossref PubMed Scopus (1851) Google Scholar), PDGF-A (21.Qin Y. Rezler E.M. Gokhale V. Sun D. Hurley L.H. Nucleic Acids Res. 2007; 35: 7698-7713Crossref PubMed Scopus (173) Google Scholar, 22.Guo K. Pourpak A. Beetz-Rogers K. Gokhale V. Sun D. Hurley L.H. J. Am. Chem. Soc. 2007; 129: 10220-10228Crossref PubMed Scopus (227) Google Scholar), RET (22.Guo K. Pourpak A. Beetz-Rogers K. Gokhale V. Sun D. Hurley L.H. J. Am. Chem. Soc. 2007; 129: 10220-10228Crossref PubMed Scopus (227) Google Scholar), and the diabetes susceptibility locus (23.Lew A. Rutter W.J. Kennedy G.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12508-12512Crossref PubMed Scopus (103) Google Scholar). In addition, a promoter region in the human insulin gene can form a quadruplex structure in vitro capable of specifically binding insulin suggesting a quadruplex based feedback expression loop (24.Connor A.C. Frederick K.A. Morgan E.J. McGown L.B. J. Am. Chem. Soc. 2006; 128: 4986-4991Crossref PubMed Scopus (83) Google Scholar). In RNA the 5′ untranslated region of a number of proto-oncogenes contain quadruplex forming motifs including NRAS, BCL2, JUN, and FGR (25.Kumari S. Bugaut A. Huppert J.L. Balasubramanian S. Nat. Chem. Biol. 2007; 3: 218-221Crossref PubMed Scopus (587) Google Scholar). In the case of NRAS,5′ untranslated region mutations that disrupt the ability of the transcript to form quadruplex structures appear to increase translation over 3-fold, suggesting that the 5′ untranslated region quadruplex has a function in inhibiting translation of certain mRNAs (25.Kumari S. Bugaut A. Huppert J.L. Balasubramanian S. Nat. Chem. Biol. 2007; 3: 218-221Crossref PubMed Scopus (587) Google Scholar). There is also evidence that preRNA termination regions have G-rich regions capable of forming quadruplex structures and possibly effect usage of alternative polyadenylation sites (26.Arhin G.K. Boots M. Bagga P.S. Milcarek C. Wilusz J. Nucleic Acids Res. 2002; 30: 1842-1850Crossref PubMed Scopus (79) Google Scholar). The predicted biological problems of blockage of replication, translation, and perhaps RNA processing through the formation of highly stable quadruplex structures led us and others to search for enzymatic quadruplex resolvases. RecQ family proteins BLM and WRN have been shown to possess DNA quadruplex resolving activity (18.Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 27.Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar) as has recently the dog-1 homolog DEXH helicase FANCJ (28.Wu Y. Shin-Ya K. Brosh Jr., R.M. Mol. Cell. Biol. 2008; 28: 4116-4128Crossref PubMed Scopus (330) Google Scholar) whose loss is associated with familial breast cancer and Fanconi anemia. We took a biochemical approach to detect and fractionate quadruplex G4-DNA resolving activity, and initially we characterized a detectable human G4-DNA resolvase activity that was NTP-dependent and free of nuclease activity (29.Harrington C. Lan Y. Akman S.A. J. Biol. Chem. 1997; 272: 24631-24636Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Subsequently, through using an affinity chromatography/proteomics approach, we identified the major tetramolecular DNA resolvase in HeLa cells as the DHX36 gene product, naming the protein G4R1 4The abbreviations used are: G4R1, G4 resolvase 1; GSPB, G4-DNA streptavidin paramagnetic beads; RHAU, DHX36 gene product from RNA helicase associated with AU-rich element of urokinase mRNA; (HHN)11, 11 repeats of a nucleotide sequence, where H = C, A, or T/U, and N = A, C, G, or T/U; TAMRA, 5,6-carboxytetramethylrhodamine; shRNA, short hairpin RNA; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. for G4 Resolvase 1 (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This protein was previously isolated for its affinity to AU-rich mRNA, possibly through interaction with the protein PARN, and termed RHAU (RNA helicase associated with AU-rich elements (31.Tran H. Schilling M. Wirbelauer C. Hess D. Nagamine Y. Mol. Cell. 2004; 13: 101-111Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar)). Therefore, we now refer to this protein by the double acronym G4R1/RHAU. Further work has shown that G4R1/RHAU can co-localize in nuclei with and co-immunoprecipitate with the RNA processing helicases p68 and p72 (32.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (42) Google Scholar). These observations led us to consider that the G4-RNA quadruplex may be a target of G4R1/RHAU activity. Logically, there should be great opportunity for quadruplex structures to form in RNA. RNA quadruplexes have been found to be more stable than their cognate DNA quadruplexes of the same sequence and structure under physiological salt conditions (33.Sacca B. Lacroix L. Mergny J.L. Nucleic Acids Res. 2005; 33: 1182-1192Crossref PubMed Scopus (208) Google Scholar). RNA molecules are largely single-stranded for extended periods of time, allowing the kinetic potential for the formation of bimolecular and tetramolecular quadruplex structures. In addition, complementary strand hybridization does not compete for single-stranded RNA as it does for DNA quadruplex structures formed from duplex DNA. In this study we demonstrate and characterize a tetramolecular RNA quadruplex resolvase activity associated with G4R1/RHAU for the first time. In addition, we determine that G4R1/RHAU has a very tight binding constant for tetramolecular quadruplex structures of both RNA and DNA. The robust resolution of RNA quadruplex by G4R1/RHAU may suggest that removal of quadruplex in RNA is an important part of RNA processing in cells. G4-Nucleic Acid Formation—Tetramolecular G4-quadruplex DNA and RNA were formed using an identical procedure and are herein referred to as "dAGA" or "rAGA." The oligonucleotides used were of the identical base sequence whether of DNA or RNA: AAAAA AAAAA AAAAA GGGGG AAAAA AAAAA AAAAA. Oligoribonucleotides (unlabeled or 5′-TAMRA4-labeled) were purchased from Dharmacon Research and oligodeoxyribonucleotides (unlabeled or 5′-TAMRA-labeled) were purchased from either Oligos Etc. or Integrated DNA Technologies. Oligodeoxyribonucleotides were dissolved at a concentration of 0.5 mm in RNase-free 10 mm Tris-HCl, 1 mm EDTA, pH 7.5. Oligoribonucleotides were deprotected according to the manufacturer's instructions and dissolved in RNase-free 10 mm Tris-HCl, 1 mm EDTA, pH 7.5, at a concentration of 0.5 mm. Oligonucleotide solutions were aliquoted into PCR tubes and incubated in a thermocycler (Eppendorf epGradient S) at 98 °C for 10 min, then held at 80 °C. Immediately the tubes were opened and EDTA, pH 8, was added to a final concentration of 25 mm. The tubes were reclosed and allowed to come slowly to room temperature. Aliquots were combined and stored at 4 °C for 2–3 days. PAGE analysis of the annealed oligonucleotides indicated that this annealing procedure resulted in over 99% conversion of monomer to tetramolecular quadruplex (data not shown). G4 nucleic acids formed from 5′-TAMRA-labeled oligonucleotides were further purified by electrophoresis on a 10% polyacrylamide gel and band excision. Electrophoretic bands containing G4 nucleic acids were recovered by electroelution in a Schleicher and Schuell Elutrap with 1× TBE buffer additionally containing 3 mm KCl. The Elutrap was run with buffer recirculation. 5′-TAMRA-labeled G4 nucleic acids were aliquoted and stored at –20 °C or lower. 5′-[32P]-End Labeling of G4 Nucleic Acids—5′-[32P]-End-labeled G4 oligonucleotides were obtained by incubating unlabeled G4 oligonucleotides annealed as described above with T4 polynucleotide kinase (Promega Corp.) and [γ-32P]ATP for 0.5 h at 37 °C, according to the manufacturer's instructions. 5′-[32P]-Labeled G4 oligonucleotides were purified with a MicroSpin G25 column (GE Healthcare) equilibrated with TEK (10 mm Tris, 1 mm EDTA, and 50 mm KCl) and stored at –20 °C. Production and Purification of Recombinant G4R1/RHAU—G4-DNA bound streptavidin paramagnetic beads (GSPB) were prepared as described previously (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). E. coli strain Rosetta 2 (Novagen) was transformed with TriEx-4 DHX36 (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) expression plasmid. Cultures were grown to a density of A600 = 0.4– 0.6 at 37 °C in a Brunswick shaker incubator. Cultures of 500 ml were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at 4 °C and grown overnight at 14 °C with gentle agitation. Recombinant G4R1/RHAU protein was initially purified by means of a His6 tag by utilizing the TALON cobalt beads and xTractor kit according to manufacturer's (Clontech) instructions with 2× Sigma protease inhibitor mixture and 15 μg/ml leupeptin added. Rosetta 2 cell lysates were isolated and bound to TALON cobalt (0.5 ml bead volume per 500 ml of E. coli culture) resin as recommended by the manufacturer. Cobalt resin was washed three times with ice-cold SSC (4 ×) with β-mercaptoethanol (0.5 μl/ml). Recombinant protein was eluted from resin with three washes of 0.5 ml of histidine elution buffer (0.7 m histidine, pH 6.0, 8.6 mm β-mercaptoethanol, 1× Sigma protease inhibitor mixture), followed by one 0.5-ml wash of 200 mm EDTA, pH 6.0. For the second phase of purification, the four eluates were combined with 1 ml (3 ml total) of 3× Res buffer (1×, 50 mm Tris acetate, pH 7.8, 50 mm NaCl, 70 mm glycine, 0.5 mm MgCl2, 0.012% bovine α-lactalbumin, 1× Sigma protease inhibitor mixture, 10% glycerol) and bound to GSPB at 37 °C for 15 min. Bound GSPB were washed two times in ice-cold SSC (4×) with 0.1% α-lactalbumin and 0.5 μl/ml β-mercaptoethanol. High purity recombinant His-tagged DHX36 protein (10,000 units/μg, units defined previously (29.Harrington C. Lan Y. Akman S.A. J. Biol. Chem. 1997; 272: 24631-24636Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar)) was obtained by ATP-dependent elution of GSPB as described previously (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), except bovine α-lactalbumin and Sigma protease inhibitor mixture were added to the elution buffer. Purified enzyme was stored at –80 °C. Electrophoretic Mobility Shift (Bandshift) Assays and Apparent Kd Determination—The apparent Kd of G4R1/RHAU bound to tetramolecular G4-DNA or -RNA was estimated by bandshift assay. Estimation of recombinant G4R1/RHAU concentration was performed on 4–12% SDS-PAGE (Invitrogen) and Coomassie Blue stain (ProtoBlue Safe, National Diagnostics). A dilution series of recombinant G4R1/RHAU was loaded next to a dilution series of protein standards (Promega, V849A). Gels were scanned on an Epson 2450 flatbed scanner using transmissive mode and Silverfast (version 6) TWAIN. Band densities were analyzed using Multi Gauge (Fuji) software. Recombinant G4R1/RHAU at concentrations of 10–300 pm was incubated with 1 pm 5′-32P-labeled G4 nucleic acid in K-Res buffer with 10 mm EDTA at 37 °C for 30 min. Binding mixtures were then loaded as is (monitored visually by Schlieren lines) and analyzed by 10% non-denaturing PAGE. Electrophoresis was performed at 70 volts for 10 h in a cold room (7 °C). Gels were imaged on a Typhoon 9210 Imager (GE Healthcare). Band densities were analyzed using Multi Gauge (Fuji) software and statistical analysis with MS Excel. Linear regression models were fit to the data points corresponding to at least 30 and 50 pm for percent bound substrate. The apparent Kd value was estimated from each model at 50% bound substrate for four experiments, separately for RNA and DNA. A two-sided t test was used to compare the average apparent Kd values for RNA and DNA. A two-sided p value <0.05 was considered to be statistically significant. G4-DNA and G4-RNA Competition Assay—Resolvase activity was determined as previously described (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) but in a modified RES buffer ("K-Res," 100 mm KCl, 10 mm NaCl, 3 mm MgCl2, 50 mm Tris acetate, pH 7.8, 70 mm glycine, 0.012% bovine α-lactalbumin, 10% glycerol). 0.2 pmol of 5′-end-labeled quadruplex (32P or TAMRA) were included per 50-μl reaction. Reactions were allowed to proceed at 37 °C for 30 min, stopped by addition of 5 μl of 200 mm EDTA, and analyzed by electrophoresis through a 10% non-denaturing polyacrylamide TBE gel with 10% glycerol. G4 nucleic acids used in competition experiments included tetramolecular dAGA and rAGA quadruplexes and E. coli tRNA (Sigma). Competitor nucleic acid concentrations ranged from a 128-fold molar excess of competitor to labeled tetramolecular quadruplex DNA substrate down to an equimolar competitor to substrate ratio. In a half-volume reaction mixture (25 μl), 2× competitor nucleic acids were mixed with 0.2 pmol of TAMRA-labeled G4 nucleic acid substrate on ice. Next, 25 μl of reaction mixture containing 1 unit of recombinant G4R1/RHAU was added (50 μl total reaction volume) at 4 °C, then reaction mixtures were incubated at 37 °C for 30 min, dropped to 4 °C, and stopped with 5 μl of 200 mm EDTA. Gels were scanned on a Typhoon 9210 Imager (GE-Healthcare) and images were analyzed using FUJI Multi Gauge version 3.0 imaging software. Statistical analysis was done in Microsoft Excel 2003 and standard deviations were calculated by the STDEV function. Development of Tetracyline Inducible shRNA for G4R1/RHAU Down-regulation—The T-REx™ HeLa cell line (pTER-RHAU25) that expresses G4R1/RHAU-targeting shRNA molecules under doxycycline control was described previously (32.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (42) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium + 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mm l-glutamine, 450 μg/ml Zeocin, and 3 μg/ml blasticidine in T-75 filter-cap culture flasks. At ∼60–70% confluency, doxycycline (1 μg/ml final) was added daily with fresh media for 5 days. Cells were scraped in cold phosphate-buffered saline, collected in 1.5-ml microtubes, and centrifuged into pellets. The pellets were frozen by storage at –80 °C. Assay of G4 Nucleic Acid Resolving Activity from HeLa Cells Expressing G4R1/RHAU shRNA—T-REx™ HeLa cell extracts were prepared as reported previously (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Briefly, cell pellets were thawed and manually homogenized on ice in an equal volume of cold 2× Lysis buffer (100 mm Tris acetate, pH 7.8, 0.4 mm EDTA, 40 mm β-mercaptoethanol, 0.02% Triton X-100, 20% glycerol, 50 μg/ml leupeptin, 1× Sigma protease inhibitor mixture) with a microtube pestle (USA Scientific, 1415–5390). Cell particulates were removed by centrifugation (Eppendorf microcentrifuge, 14 krpm for 5 min). Whole cell extracts were aliquoted and stored at –80 °C. Extracts were assayed for G4 nucleic acid resolving activity as described above in K-Res buffer supplemented with 1/100th reaction volume of SUPERase-In™ (Ambion) and 0.5 mm MgCl2, using 5′-TAMRA-labeled G4 nucleic acid substrates. Western Blotting of HeLa Cell Extracts—Western blotting was done by standard methods. Briefly, 10 μg each of HeLa pTER-RHAU25 whole cell extracts were resolved by SDS-PAGE on 4–12% BisTris Invitrogen gradient gels and transferred to Hybond P (polyvinylidene difluoride, Amersham Biosciences). Blots were blocked with 5% (Carnation Instant Nonfat Dry) milk, TBST (0.1% Tween) and probed with RHAU monoclonal antibody 12F33 (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) at 1:15,000 dilution in 2% milk TBST and β-actin monoclonal antibody at 1:10,000 dilution (Sigma A5441) in 5% milk TBST. Bio-Rad goat anti-mouse IgG (H+L)-horseradish peroxidase conjugate was used as the secondary antibody at concentrations ranging from 1:7,500 to 1:10,000 in 2–5% milk in TBST. Membranes were activated for chemiluminescence by ECL-plus (Amersham Biosciences) and exposed to Kodak Biomax Light film. Films were scanned on an Epson (2450) flatbed scanner using transmissive mode and Silverfast (version 6) twain. Band densities were measured using Multi Gauge (Fuji) software and data analyzed with MS Excel. G4R1/RHAU Displays Robust Resolving Activity on Both RNA and DNA Tetramolecular Quadruplex Substrates—Our previous work showed that recombinant G4R1/RHAU protein resolves tetramolecular DNA quadruplex efficiently and specifically (30.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). We have previously defined 1 unit of G4R1 resolvase activity as that amount that resolves 50% of 0.2 pmol of tetramolecular quadruplex DNA in 30 min at 37 °C in 30 μl of reaction buffer (29.Harrington C. Lan Y. Akman S.A. J. Biol. Chem. 1997; 272: 24631-24636Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Fig. 1 compares resolution of both RNA and DNA tetramolecular quadruplex substrates for a range of G4R1/RHAU concentrations under standard reaction conditions. Nondenaturing gel electrophoresis of reaction contents demonstrated G4R1/RHAU concentration-dependent resolution of both RNA and DNA quadruplex structures into single strands. At concentrations of greater than 1 unit/reaction, G4R1/RHAU catalyzed complete resolution of both RNA and DNA tetramolecular quadruplex substrates 5′-end-labeled with either [32P] (Fig. 1, A and C) or the fluorescent molecule TAMRA (Fig. 1, B and D) into single strands. One unit of G4R1/RHAU resolved slightly more 32P-end-labeled quadruplex DNA than quadruplex RNA substrate (compare Fig. 1A, lane 4 with C, lane 4), whereas 1 unit of G4R1/RHAU resolved a similar amount of TAMRA-labeled quadruplex DNA and RNA (see Fig. 1, B lane 3 and D, lane 3). G4R1/RHAU concentrations less than 1 unit/reaction catalyzed less than 50% resolution of tetramolecular DNA or RNA quadruplex substrates as expected. These data demonstrate that G4R1/RHAU has robust resolving activity upon RNA quadruplex substrates in addition to DNA quadruplex substrates. Moreover, these data indicate that there is little difference in G4R1/RHAU-catalyzed resolving activity on tetramolecular quadruplex RNA and DNA substrates under these experimental conditions. G4R1/RHAU Displays a Tight Binding Affinity for Both DNA Quadruplex and RNA Quadruplex—The ability of G4R1/RHAU to bind quadruplex RNA and DNA structures was measured by gel mobility s

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