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Originally published In Press as doi:10.1074/jbc.M401752200 on July 19, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40405-40411, September 24, 2004
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Chain Termination and Inhibition of Saccharomyces cerevisiae Poly(A) Polymerase by C-8-modified ATP Analogs*{boxs}

Lisa S. Chen and Terry L. Sheppard{ddagger}

From the Department of Chemistry and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois 60208-3113

Received for publication, February 17, 2004 , and in revised form, June 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) toward various C-2- and C-8-modified ATP analogs was examined. 32P-Radiolabeled RNA oligonucleotide primers were incubated with yPAP in the absence of ATP to assay polyadenylation using unnatural ATP substrates. The C-2-modified ATP analogs 2-amino-ATP and 2-chloro (Cl)-ATP were excellent substrates for yPAP. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all produced chain termination of polyadenylation, and no primer extension was observed with the C-8-halogenated derivatives 8-Br-ATP and 8-Cl-ATP. The effects of modified ATP analogs on ATP-dependent poly(A) tail synthesis by yPAP were also examined. Whereas C-2 substitution (2-amino-ATP and 2-Cl-ATP) had little effect on poly(A) tail length, C-8 substitution produced moderate (8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) to substantial (8-Br-ATP and 8-Cl-ATP) reduction in poly(A) tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA primers, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was prepared. Polyadenylation of this modified RNA primer by yPAP in the presence of ATP was blocked completely. To probe potential mechanisms of inhibition, two-dimensional NMR spectroscopy experiments were used to examine the conformation of two C-8-modified AMP nucleotides, 8-Cl-AMP and 8-amino-AMP. C-8 substitution in adenosine analogs shifted the ribose sugar pucker equilibrium to favor the DNA-like C-2'-endo form over the C-3'-endo (RNA-like) conformation, which suggests a potential mechanism for polyadenylation inhibition and chain termination. Base-modified ATP analogs may exert their biological effects through polyadenylation inhibition and thus may provide useful tools for investigating polyadenylation biochemistry within cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modified nucleoside analogs have been used extensively as chemotherapeutic agents (1). These analogs of cellular nucleosides are based on structural alterations of the ribose sugar ring, as with 3'-azido-3'-deoxyazidothymidine (2) and acyclovir (3), or modifications of the purine or pyrimidine base, such as fludarabine (9-{beta}-D-ribofuranosyl-(2-fluoroadenine) monophosphate) (4) and 2-chlorodeoxyadenosine (5). Nucleoside antimetabolites typically are taken up by cells, metabolized, and subsequently enter cellular nucleotide pools, where they exert their chemotherapeutic action. In particular, modified purine analogs have demonstrated promise for cancer treatment. Our biochemical studies have centered on two C-8-modified adenosine analogs, 8-chloroadenosine (8-Cl-Ado)1 and 8-aminoadenosine (8-amino-Ado), which have demonstrated antineoplastic activity (6, 7) and are under investigation for application to multiple myeloma and leukemia treatments.

8-Cl-Ado and 8-amino-Ado recently have been shown to induce apoptosis in cancer cell lines in culture (6, 7).2 Analysis of the cellular metabolism of 8-Cl-Ado and 8-amino-Ado revealed that treated cells rapidly accumulate the 5'-phosphorylated derivatives, 8-Cl-ATP and 8-amino-ATP, respectively, which appear to be the cytotoxic agents (6, 9).2 In both cases, a concurrent decrease in endogenous ATP is observed. Further analysis of 8-Cl-Ado cellular metabolism indicated that the effects of 8-Cl-Ado appear to be RNA-directed; treated cells exhibit decreased total cellular RNA levels and show 8-Cl-AMP incorporation into RNA (10). Apoptosis induction by C-8-modified adenosine analogs may arise from parallel changes in cellular biochemistry, including alteration of cellular ATP levels and interference with biosynthetic pathways. Incorporation of C-8-modified analogs into RNA polymers may inhibit enzymatic chain extension during transcription or polyadenylation and/or block subsequent RNA processing and transport. We previously showed that 8-Cl-Ado incorporation into RNA duplexes leads to a decrease in RNA duplex stability of ~5 kcal/mol/8-Cl-Ado modification, despite the preference of 8-Cl-Ado to maintain base pairing affinity for uracil over other nucleobases (11). The corresponding deoxynucleoside analog, 8-Cl-dAdo, also maintains standard base pairing during enzymatic DNA synthesis by the Klenow fragment of DNA polymerase I (12). However, the efficiency of DNA synthesis is diminished when 8-Cl-dAdo serves as a template base or is incorporated as an incoming nucleotide (12). These studies suggest that 8-Cl-Ado metabolites may display potent effects on the synthesis and stability of cellular nucleic acids. Given the RNA-directed nature of 8-Cl-Ado action, our research efforts have been focused on the biochemical effects of 8-modified adenosine analogs on RNA synthesis and processing.

Treatment of multiple myeloma cells with 8-Cl-Ado results in a rapid reduction in total RNA levels. Because cellular RNA levels result from a balance of biosynthesis and RNA degradation, 8-modified adenosine analogs may exert their effects by inhibition of transcription as well as acceleration of RNA degradation. Although previous studies have shown transcriptional incorporation of 8-modified adenosine derivatives into RNA polymers (10), we reasoned that 8-Cl-AMP incorporation may affect downstream RNA processing or transport steps required for mRNA maturation. Furthermore, because of their structural similarities to ATP, the analogs may inhibit the activities of other ATP-dependent RNA polymerases, such as poly(A) polymerase. Interference with RNA processing provides a potential mechanism to enhance the degradation of RNA transcripts and may explain the phenotype of cells treated with C-8-modified adenosine analogs.

Inhibition of cleavage and polyadenylation events may be a potential mechanism for the observed decrease in cellular RNA levels following 8-Cl-Ado and 8-amino-Ado treatment. Processing of pre-mRNA into mature mRNA involves three major steps: 5'-end methylguanosine capping, 3'-end cleavage and polyadenylation, and intron splicing (13). Cleavage and polyadenylation in vivo are a tightly regulated process coupled to transcription (14), involving numerous protein complexes (15, 16). RNA poly(A) tail length has been shown to regulate RNA stability (17), and thus, polyadenylation inhibition may contribute to the decreased RNA levels observed upon treatment of cells with 8-Cl-Ado. In addition to increased RNA degradation, the consequences of polyadenylation inhibition in eukaryotic systems include impaired mRNA transport from the nucleus and altered translation efficiency (18). Known RNA processing inhibitors include other ATP analogs, such as cordycepin triphosphate (3'-dATP), an inhibitor of transcription and polyadenylation (19, 20), and Ara-ATP, an inhibitor of cleavage and polyadenylation (21–23).

Enzymatic assays using poly(A) polymerase from Saccharomyces cerevisiae (yPAP) frequently have served as a biochemical model system for polyadenylation in cells (24). Earlier studies examined the substrate specificity of several ribonucleotide, deoxyribonucleotide, and dideoxyribonucleotide triphosphate analogs (25, 26) with purified yPAP. However, the inhibitory properties of C-8-modified adenosine triphosphates were not evaluated. Thus, assays of C-8-modified ATP analogs in polyadenylation reactions may provide insight into the mechanism of cellular RNA depletion in cells treated with C-8-modified purine nucleoside analogs. Furthermore, if these analogs demonstrate selective inhibition of polyadenylation enzymes, they may be useful reagents for dissecting biochemical steps in polyadenylation pathways in cells.

In the studies described herein, we examined the substrate specificity of yPAP for modified ATP derivatives that may be useful for treatment of hematological malignancies. We chose yPAP as a cell-free polyadenylation model system to provide an initial evaluation of the effects of C-8-modified ATP analogs on RNA polyadenylation. First, we assessed the inhibitory effects of various modifications at C-8 of ATP and compared them with analogous modifications at the C-2 site of the base. Modified ATP analogs were also assayed as substrate analogs and as potential inhibitors of polyadenylation in the presence of ATP. We observed that C-2-modified ATP analogs are substrates for yPAP and that C-8-modified ATP analogs are chain terminators and/or inhibitors of polyadenylation. Polyadenylation of an 8-Cl-Ado-modified RNA primer was also examined, and 3'-terminal 8-Cl-AMP residues abrogated polyadenylation by yPAP in the presence of ATP. Polyadenylation inhibition by 8-modified adenosine analogs may be due to a conformational change imposed by C-8 substitution. The conformations of C-8-modified AMP analogs were examined using NMR spectroscopy as models of the chain-terminated 3'-ends of pre-mRNA. Our model studies suggest that C-8 substitution shifts the sugar pucker equilibrium in aqueous solution to favor the DNA-like C-2'-endo form over the RNA-like C-3'-endo conformer. Perturbation of the ribose ring conformation may alter the substrate efficiency of modified ATP analogs with yPAP and the structure of the 3'-end of pre-mRNA substrates. Furthermore, C-8-modified ATP analogs, which inhibit poly(A) polymerase via several mechanisms, may be useful for the dissection of individual biochemical steps in polyadenylation pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All reactions were performed in oven-dried (140 °C) glassware using anhydrous Aldrich Sure-SealTM solvents and N2 as an inert atmosphere. Unless otherwise noted, reagents were obtained from Aldrich and used without further purification. Tributylamine was freshly distilled and stored over 4-Å molecular sieves. DEAE-Sephadex A-25 resin (chloride counterion) was used for ion exchange chromatography. The resins were soaked in 1 M triethylammonium bicarbonate buffer (pH 8.0) and then swelled in deionized water prior to use. 31P NMR data were recorded at 162 MHz on a Varian 400 spectrometer and referenced to an external standard of 85% aqueous phosphoric acid. RNA oligonucleotides were analyzed by RP-HPLC with a Waters Prep 600 HPLC using an acetonitrile gradient in 0.1 M triethylammonium acetate buffer (pH 7.2). RNA oligonucleotides were characterized using a Voyager-DETM PRO BiospectrometryTM workstation MALDI-TOF mass spectrometer (PerSeptive Biosystems, Inc., Foster City, CA). An N2 laser was used (337-nm wavelength, 3-ns pulse), and spectra were acquired in the linear positive or negative ion mode, averaging 128 shots. A MALDI-TOF-MS matrix was prepared by combining an 8:1 ratio of 0.2 M 2,4,6-trihydroxyacetophenone in 50% CH3CN/H2O and 0.3 M ammonium citrate (H2O). (dT)10 and (dT)18 sequences (Integrated DNA Technologies) were used as internal standards for calibration of molecular masses in MALDI-TOF-MS experiments. Samples were prepared using a Millipore C-18 ZipTipTM to remove excess salts. T4 polynucleotide kinase (30 units/µl) and yPAP (600 and 815 units/µl) were purchased from United States Biochemical Corp. Enzyme concentrations of yPAP for polyadenylation assays (reported in units/µl of reaction total volume in the protocols below) corresponded to 10–16 nM for each experiment. A 10-bp DNA ladder was obtained from Invitrogen. NAP-5 Sephadex G-25 columns were obtained from Amersham Biosciences, and [{gamma}-32P]ATP (7000 Ci/mmol) was purchased from ICN. Radioactive bands on polyacrylamide gels were visualized using an Amersham Biosciences Storm PhosphorImager and analyzed using Amersham Biosciences ImageQuant software.

ATP Derivatives—ATP was obtained from Amersham Biosciences, and 2-Cl-ATP was obtained from Ambion Inc. 2-Amino-ATP, 8-azido-ATP, and 8-aza-ATP were purchased from TriLink Biotechnologies, and 8-Br-ATP was obtained from Sigma. 8-Cl-Ado (see Scheme 1, 1) was synthesized as described (11), and 8-amino-Ado (2) was synthesized from 8-bromoadenosine (27).



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  		SCHEME 1.
Chemical phosphorylation of 8-modified adenosine derivatives. DMF, N,N-dimethylformamide; Bu, butyl.

 
8-Cl-AMP (3) and 8-Amino-AMP (5)—To a stirred solution of 1 (0.030 g, 0.10 mmol) or pink emulsion of 2 (0.028 g, 0.10 mmol), dissolved in 0.25 ml of trimethyl phosphate, was added phosphorus oxychloride (0.018 ml, 0.2 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and then quenched with 2 ml of 1.0 M triethylammonium bicarbonate buffer and concentrated in vacuo. The product was purified by DEAE-Sephadex ion exchange chromatography (0–1.0 M triethylammonium bicarbonate), lyophilized, and dissolved in 2 ml of methanol. A 1.0 M NaI solution in acetone (5 ml) was added; the resulting slurry was centrifuged; and the supernatant was removed. The precipitate was rinsed with acetone (2 x 5 ml) by centrifugation, and the solvent was removed to give 0.032 g of white powder (3, 78%) or 0.011 g of pale pink powder (5, 14%). 3: 31P NMR (D2O); {delta} 2.78; ESI-MS, calculated [M]– = 380.0 and found [M]– = 380.1. 5: 31P NMR (D2O); {delta} 0.64; ESI-MS, calculated [M]– = 361.1 and found [M]– = 361.1.

8-Cl-ATP (4) and 8-Amino-ATP (6)—To a stirred solution of 1 (0.030 g, 0.10 mmol) or pink emulsion of 2 (0.028 g, 0.10 mmol), dissolved in 0.25 ml of trimethyl phosphate, was added phosphorus oxychloride (0.018 ml, 0.2 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, and then 0.1 ml of tributylamine and a solution of ammonium pyrophosphate (0.228 g, 0.50 mmol) in 1 ml of anhydrous N,N-dimethylformamide was added. The reaction mixture was stirred at 0 °C for 5 min, quenched with 5 ml of 1.0 M triethylammonium bicarbonate buffer, and concentrated in vacuo. The product was purified by DEAE-Sephadex ion exchange chromatography (0–1.0 M triethylammonium bicarbonate), lyophilized, and dissolved in 2 ml of methanol. A 1.0 M NaI solution in acetone (5 ml) was added; the resulting slurry was centrifuged; and the supernatant was removed. The white precipitate was rinsed with acetone (2 x 5 ml) by centrifugation, and the solvent was removed to give 0.053 g of white powder (4, 90%) or 0.007 g of pale pink powder (6, 13%). 4: 31P NMR (D2O); {delta} –7.50, –10.17, and –21.50; ESI-MS, calculated [M]– = 540.0 and found [M]– = 540.0. 6: 31P NMR (D2O); {delta} –4.93, –10.56, and –20.34; ESI-MS, calculated [M]– = 521.2 and found [M]– = 521.1.

RNA Oligonucleotides—RNA primer 5'-UGUGCCCGA-3' (7) was purchased from Dharmacon Research Inc. and was deprotected at 37 °C for 30 min with 400 µl of 100 mM acetic acid, adjusted to pH 3.8 with TEMED. The purity was verified by RP-HPLC and MALDI-TOF-MS (calculated [M]+ = 2832.8 and found [M]+ = 2829.7), and the oligonucleotide was used without further purification. Oligonucleotide 5'-UGUGCCCGACl-3' (8) was synthesized using an 8-Cl-Ado-derivatized Controlled-Pore Glass support as described previously (11), purified by RP-HPLC, and characterized by MALDI-TOF-MS (calculated [M]– = 2865.2 and found [M]– = 2865.6).

Preparation of 5'-32P-Radiolabeled Oligonucleotides—RNA oligonucleotide 7 or 8 (20 pmol) was incubated at 37 °C for 60 min with 10.5 units of T4 polynucleotide kinase in a 20-µl reaction mixture containing 10 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, and 24 pmol of [{gamma}-32P]ATP (8.4 Ci/µl, 7000 Ci/mmol). The reaction was quenched with 2 µl of 100 mM EDTA, and the labeled DNA was purified by 7 M -20% dPAGE with 89 mM Tris, 89 mM borate, and 1mM EDTA as the running buffer. The radiolabeled oligonucleotide was visualized by autoradiography, excised, and eluted by soaking the gel piece in 100 µl of buffer (200 mM NaCl, 10 mM Tris (pH 7.5), and 1 mM EDTA) at 25 °C for 12 h. The eluted product was purified using a NAP-5 Sephadex G-25 column and stored in double-distilled H2O.

yPAP Substrate Specificity Assays—Radiolabeled RNA oligonucleotide 7 was incubated at 37 °C for 1 h in a solution containing yPAP and a single modified nucleoside triphosphate in the absence of ATP. The extension reactions were analyzed by 7 M-20% dPAGE. Final extension conditions were as follows: 100 µM NTP, 200 nM primer 7, 1.25 units/µl yPAP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol. The triphosphates assayed were ATP, 3'-dATP, 2-Cl-ATP, 2-amino-ATP, 8-Br-ATP, 8-Cl-ATP, 8-amino-ATP, 8-azido-ATP, and 8-aza-ATP.

Isolation and Characterization of Chain Termination Products— RNA primer 7 was incubated at 37 °C for 1 h in a solution containing 15 µM primer 7, 1.0 mM 3'-dATP or 1.15 mM 8-amino-ATP, 4 units/µl yPAP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol. To remove proteins from the reaction mixture, the reactions were extracted with 20 µl of 25:24:1 phenol/chloroform/isoamyl alcohol (pH 8.0). The phenol layers were back-extracted with 20 µl of Tris-EDTA buffer (pH 8.0), and the combined aqueous layers were extracted with 40 µl of 24:1 chloroform/isoamyl alcohol to remove residual phenol. The products were analyzed by RP-HPLC using a Waters XTerraTM RP18 column (C-18, 3.5 µm, 4.6 x 50 mm) and a 5–10% CH3CN gradient in 0.1 M triethylammonium acetate buffer (pH 7.2) for 40 min. Unmodified 7 eluted at tR = 16 min, and products from both 3'-dATP and 8-amino-ATP extension reactions eluted at tR = 22 min. The products were frozen, lyophilized, and desalted using a C-18 ZipTipTM. MALDI-TOF-MS (m/z): 3'-dAMP termination product (5'-UGUGCCCGAdA-3'), calculated [M]+ = 3145.0 and found [M]+ = 3144.5; 8-amino-AMP termination product (5'-UGUGCCCGAANH2-3'), calculated [M]+ = 3177.0 and found [M]+ = 3177.8.

Evaluation of Kinetic Parameters (Km) for ATP Analogs 3'-dATP and 8-Amino-ATP—Radiolabeled RNA oligonucleotide 7 (200 nM) was incubated at 37 °C for 20 min using single hit completed conditions (28) in a solution containing 4–50 µM 3'-dATP or 1–100 µM 8-amino-ATP, 1.1 units/µl yPAP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol. The reaction was initiated by the addition of analog triphosphate. Aliquots were removed during the reaction, and the extension reactions were terminated by adding 1 volume of 2x gel loading buffer (20% (w/v) sucrose, 0.05% bromphenol blue, 0.05% xylene cyanol FF, 0.1% SDS, 16 M urea, 89 mM Tris, 89 mM borate, and 1 mM EDTA). Each reaction was analyzed by 7 M-20% dPAGE. The ratio of terminated product to unextended primer was determined from the relative band intensities of the imaged gel. Extension data were analyzed using Lineweaver-Burk double-reciprocal plots, and the observed kinetic parameters were extracted using linear regression analysis of the plots. Data were also analyzed by fitting the Michaelis-Menten expression, which gave Km values within 20% of the Lineweaver-Burk values.

Polyadenylation Inhibition Assays with 8-Halogenated ATP—Radiolabeled RNA oligonucleotide 7 (200 nM) was incubated at 37 °C for 1 h in a solution containing 1.1 units/µl yPAP, 100 µM ATP, 25–100 µM 8-Br-ATP or 8-Cl-ATP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol. The extension reactions were terminated by the addition of 1 volume of 2x gel loading buffer, and the products were analyzed by 7 M-20% dPAGE.

Polyadenylation Inhibition Assays with Unnatural Triphosphates and ATP—Radiolabeled RNA oligonucleotide 7 (200 nM) was incubated at 37 °C for 1 h in a solution containing 100 µM ATP, 50–100 µM NTP, 1.1 units/µl yPAP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol. The triphosphates assayed were ATP, 3'-dATP, 2-Cl-ATP, 2-amino-ATP, 8-Cl-ATP, 8-amino-ATP, 8-azido-ATP, and 8-aza-ATP. The extension reactions were terminated by the addition of 1 volume of 2x gel loading buffer, and the products were analyzed by 7 M-20% dPAGE.

Polyadenylation of 3'-Terminated 8-Cl-AMP Primer 8—Radiolabeled RNA primer 8 (200 nM) was incubated at 37 °C for 30 min in a solution containing 1.25 units/µl yPAP, 200 µM ATP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol. Aliquots were removed at 1, 5, 15, and 30 min, and the products were analyzed by 7 M-20% dPAGE. As a control, primer 7 was assayed under the same conditions.

NMR Conformational Analysis of Phosphorylated AMP Derivatives— AMP and AMP derivatives were dissolved in D2O (99.96 atom % D) and lyophilized three times. The samples were redissolved in 0.4–0.8 ml of D2O (99.96 atom % D), with a final sample concentration from 0.5 and 1.0 mM, then transferred to a 5-mm NMR tube. NMR data were acquired on an Varian spectrometer operating at a frequency of 600 MHz and a probe temperature of 298 K. Two-dimensional total correlation spectroscopy and DQF-COSY data were acquired in phase-sensitive mode. The digital resolution of the processed DQF-COSY spectrum was 2.34 and 15 Hz/point in the w2 and w1 dimensions, respectively. The 3JH-1',H-2' coupling constants were obtained from the splitting observed in the DQF-COSY cross-peaks in the w2 dimension. The fractional populations of the C-2'-endo conformation were obtained using the formula [C-2'-endo] = 3JH-1',H-2'/(JH-1',H-2' + 3JH-3',H-4'), where the value of 3J3H-1',H-2' + 3JH-3',H-4' is assumed to be equal to 10 Hz (29).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Synthesis of 5'-Phosphorylated 8-Modified Derivatives—The AMP and ATP derivatives 8-Cl-AMP (3) and 8-Cl-ATP (4) and 8-amino-AMP (5) and 8-amino-ATP (6) were synthesized from 8-Cl-Ado and 8-amino-Ado, respectively (30). Triphosphate derivatives 4 and 6 were synthesized in a one-pot reaction using phosphorus oxychloride and tributylammonium pyrophosphate as shown in Scheme 1. The triphosphate analogs were converted into sodium salts and isolated by RP-HPLC.

Poly(A) Polymerase Nucleotide Specificity—To assess the substrate specificity of yPAP, a number of C-2-modified (2-Cl-ATP and 2-amino-ATP) and C-8-modified (8-Br-ATP, 8-Cl-ATP, 8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) analogs were assayed with yPAP under standard conditions (24). Primer extensions with yPAP were conducted using a synthetic RNA primer (5'-UGUGCCCGA-3', 7) in reactions containing 200 nM primer 7 and 100 µM each ATP analog in the absence of ATP. A known yPAP chain terminator, 3'-dATP, was used as a positive control (31). The extension reactions were incubated at 37 °C for 1 h and then analyzed by 20% dPAGE as shown in Fig. 1. Polyadenylation in the presence of the natural substrate ATP produced a poly(A) tail several hundred nucleotides in length (Fig. 1, lane 3). Incubation with 3'-dATP, a known polyadenylation inhibitor, produced a single extension product (Fig. 1, lane 4), consistent with chain termination. Primer extension reactions with C-2- and C-8-modified nucleotides exhibited trends that could be grouped based on the type of modification. Incubation of yPAP with both C-2-modified ATP analogs produced polyadenylated product beyond a single AMP insertion (Fig. 1, lanes 5 and 6). Polyadenylation with 2-amino-ATP proceeded as efficiently as with ATP, producing a poly(A) tail several hundred nucleotides in length (Fig. 1, lane 6). Polyadenylated product was observed with 2-Cl-ATP, with, however, a decreased efficiency relative to ATP and 2-amino-ATP (Fig. 1, lane 5). yPAP was unable to processively synthesize poly(A) tails using C-8-modified ATP analogs, in contrast to C-2-modified ATP analogs. The C-8-substituted analogs were divided into two groups containing halogen or nitrogenous modifications. The halogenated derivatives, 8-Br-ATP and 8-Cl-ATP, do not appear to be substrates for yPAP, and no significant extension was detected with either analog (Fig. 1, lanes 7 and 8). The latter group, consisting of 8-amino-ATP, 8-azido-ATP, and 8-aza-ATP, all terminated polyadenylation following the incorporation of a single unnatural residue (Fig. 1, lanes 9–11).



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  		FIG. 1.
Substrate specificity of yPAP toward C-2- and C-8-modified ATP analogs. Shown is the primer elongation of RNA primer 7. A solution (5 µl) containing 200 nM 5'-32P-radiolabeled RNA primer 7, 6.25 units/µl yPAP, 100 µM nucleoside triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1, radiolabeled 10-bp DNA ladder; lane 2, 5'-32P-radiolabeled primer 7 (no NTP); lane 3, ATP; lane 4, 3'-dATP; lane 5, 2-Cl-ATP; lane 6, 2-amino-ATP; lane 7, 8-Br-ATP; lane 8, 8-Cl-ATP; lane 9, 8-amino-ATP; lane 10, 8-azido-ATP (8-N3-ATP); lane 11, 8-aza-ATP. nt, nucleotides.

 
Characterization of Terminated Products and Determination of Kinetic Parameters—To characterize the chain termination products, primer 7 (500–1000 pmol) was incubated with ~1 mM 8-amino-ATP or 3'-dATP. The termination products were separated from unextended primer 7 by RP-HPLC and analyzed by MALDI-TOF-MS. The masses obtained for the chain termination products were in excellent agreement with calculated masses: 3'-dAMP-terminated (5'-UGUGCCCGAdA-3'), calculated mass = 3145.0 and found mass = 3144.5; and 8-amino-AMP-terminated (5'-UGUGCCCGAANH2-3'), calculated mass = 3177.0 and found mass = 3177.8.

8-Amino-ATP was selected as a representative C-8-modified chain terminator for further analysis. Michaelis-Menten kinetic parameters for chain termination of polyadenylation by 8-amino-ATP were measured, and the resulting values were compared with those of a known polyadenylation chain terminator, 3'-dATP. The kinetic parameters for 8-amino-ATP and 3'-dATP were determined using a fixed concentration of primer 7, yPAP, and varying amounts of triphosphate. The measured initial velocity (V0) of primer extension was analyzed as a function of triphosphate concentration (28). The measured Michaelis constants (Km) were 1.9 µM for 8-amino-ATP and 6.3 µM for 3'-dATP as determined from Lineweaver-Burk double-reciprocal plots (Supplemental Data). The reported Km value for ATP with yPAP is 40–100 µM (32–34), suggesting that 8-amino-ATP may be an effective chain terminator during polyadenylation.

Inhibition of Polyadenylation by Halogenated C-8-modified ATP Analogs—Although 8-Br-ATP and 8-Cl-ATP were not substrates for yPAP, in light of the cellular triphosphate accumulation data, we examined the effects of halogenated ATP analogs on polyadenylation in the presence of ATP. Primer 7 was incubated at 37 °C with yPAP, 100 µM ATP, and varying amounts of 8-Br-ATP or 8-Cl-ATP, and the extension products were analyzed by 20% dPAGE (Fig. 2). Increasing the concentration of halogenated ATP analog relative to ATP resulted in a dramatic decrease in tail length. Inclusion of 25–75 µM 8-Br-ATP in the polyadenylation assay decreased the overall poly(A) tail length in a concentration-dependent manner. At equimolar concentrations of ATP and 8-Br-ATP (100 µM), the observed poly(A) tail was ~20 nucleotides or less in length (Fig. 2, lanes 4–7). The effect was more pronounced with 8-Cl-ATP, where the poly(A) tail length was shortened to <10 nucleotides at 25 µM 8-Cl-ATP (Fig. 2, lanes 8–11). The effects of 8-Cl-ATP on polyadenylation were significant at 25 µM, which is considerably lower than the observed cellular concentration of 400 µM for 8-Cl-ATP after 12 h (6). These results suggest that 8-Cl-ATP may have pronounced effects on poly(A) tail synthesis by yPAP.



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  		FIG. 2.
Reduction of poly(A) tail length by C-8-halogenated ATP analogs. Shown is the gel electrophoretic analysis of RNA primer 7 with yPAP, 100 µM ATP, and increasing amounts of 8-halogenated ATP. A solution (5 µl) containing 200 nM 5'-32P-radiolabeled RNA primer 7, 5.5 units/µl yPAP, 100 µM ATP, 25–100 µM 8-modified ATP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1, radiolabeled 10-bp DNA ladder; lane 2, 5'-32P-radiolabeled primer 7 (no ATP); lane 3, ATP only; lanes 4–7, 25, 50, 75, and 100 µM 8-Br-ATP, respectively; lanes 8–11, 25, 50, 75, and 100 µM 8-Cl-ATP, respectively. nt, nucleotides.

 
To compare the effects of 8-Cl-ATP relative to other ATP analogs, polyadenylation assays were conducted with the same panel of C-2- and C-8-modified triphosphate analogs in the presence of ATP. RNA primer 7 was incubated at 37 °C with yPAP, 100 µM ATP, and either 50 or 100 µM modified triphosphate. The reaction products were analyzed by 20% dPAGE (Fig. 3). Extension of primer 7 with ATP alone is shown in lane 3. Compared with other modified ATP analogs, 8-Cl-ATP was the most potent inhibitor of yPAP (Fig. 3, lanes 10 and 11). Primer extension reactions containing 3'-dATP produced shorter poly(A) tails (Fig. 3, lanes 4 and 5) as expected, and there was little effect observed with 2-amino-ATP (lanes 8 and 9). 2-Cl-ATP produced a moderate reduction in poly(A) tail length (Fig. 3, lanes 6 and 7), but the extension profile was similar to that of 2-Cl-ATP alone (Fig. 1). 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all resulted in a slight reduction in tail length at 50 µM, and a further reduction was observed at 100 µM. Although the C-8 nitrogen-substituted ATP derivatives appear to be moderate inhibitors of polyadenylation, the effects were less striking than with 8-Cl-ATP.



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  		FIG. 3.
Reduction of ATP-dependent poly(A) tail length by C-8-modified ATP derivatives. Shown is the gel electrophoretic analysis of RNA primer 7 extension with yPAP, 100 µM ATP, and increasing amounts of ATP analogs. A solution (5 µl) containing 200 nM 5'-32P-radiolabeled RNA primer 7, 5.5 units/µl yPAP, 100 µM ATP, 50 or 100 µM ATP analog, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1, radiolabeled 10-bp DNA ladder; lane 2, 5'-32P-radiolabeled primer 7 (no ATP); lane 3, ATP only; lanes 4 and 5, 50 and 100 µM 3'-dATP, respectively; lanes 6 and 7, 50 and 100 µM 2-Cl-ATP, respectively; lanes 8 and 9, 50 and 100 µM 2-amino-ATP, respectively; lanes 10 and 11, 50 and 100 µM 8-Cl-ATP, respectively; lanes 12 and 13, 50 and 100 µM 8-amino-ATP, respectively; lanes 14 and 15, 50 and 100 µM 8-azido-ATP, respectively; lanes 16 and 17, 50 and 100 µM 8-aza-ATP, respectively. nt, nucleotides.

 
3'-Terminal 8-Cl-AMP Residues Block Polyadenylation—The inhibition of polyadenylation with 8-Cl-ATP prompted us to address the effects of 8-Cl-Ado incorporation into RNA oligonucleotides. As stated previously, multiple myeloma cells treated with 8-Cl-Ado show incorporation of 8-Cl-AMP residues into cellular mRNA. Although we have demonstrated that 8-Cl-ATP is not a substrate for yPAP, 8-Cl-AMP incorporation into RNA during transcription may affect polyadenylation efficiency by preventing cleavage or polyadenylation of pre-mRNAs. To determine the effects of 8-Cl-AMP incorporation at 3'-terminal sites, a synthetic RNA oligonucleotide containing 3'-terminal 8-Cl-AMP was synthesized using an 8-Cl-Ado-derivatized solid support (11). Modified primer 8 (5'-UGUGCCCGACl-3') was based on the same RNA sequence as primer 7 and assayed as a polyadenylation substrate with yPAP and 200 µM ATP. Primer 7 served as a control RNA substrate. Incubation of primer 7 at 37 °C with 0.2 mM ATP and yPAP resulted in the addition of several hundred AMP residues (Fig. 4A). 8-Cl-AMP-modified RNA primer 8 was incubated under identical conditions and analyzed by 20% dPAGE. Interestingly, the site-specific introduction of 3'-terminal 8-Cl-AMP completely blocked polyadenylation by yPAP. No detectable extension of primer 8 was observed with yPAP and ATP (Fig. 4B).



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  		FIG. 4.
3'-Terminal 8-Cl-AMP residues block chain extension of RNA primers by yPAP. Shown is the gel electrophoretic analysis of RNA primer extension of unmodified primer 7 (A) and 8-Cl-AMP-terminated primer 8 with yPAP and ATP (B). A solution (20 µl) containing 200 nM 5'-32P-radiolabeled RNA primer 7 or 8, 15 units/µl yPAP, 200 µM ATP, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 µg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 30 min. For each experiment, aliquots were removed at 1, 5, 15, and 30 min, and the products were analyzed by 20% dPAGE. Lane 1, radiolabeled 10-bp DNA ladder; lane 2, 5'-32P-radiolabeled RNA primer (no ATP); lanes 3–6, extension time course at 1, 5, 15, and 30 min for each primer, respectively. nt, nucleotides.

 
NMR Analysis of C-8-modified AMP Derivatives—To gain insight into the structural basis for polyadenylation inhibition, two-dimensional NMR spectroscopy was used to evaluate the conformational preferences of 8-modified adenosine analogs in aqueous solution. Monophosphate derivatives 3 and 5 were prepared as shown in Scheme 1 and used for the NMR measurements. Experiments were performed on AMP, 8-Cl-AMP, and 8-amino-AMP in D2O. Proton assignments were obtained by analysis of the total correlation spectroscopy spectra. The three-bond scalar couplings, 3JH-1',H-2', between the H-1' and H-2' ribose protons, were obtained from cross-peaks observed in DQF-COSY spectra. The sugar pucker conformation, which was assumed to populate the C-2'-endo and C-3'-endo states (29), was determined from the 3JH-1',H-2' values, which were 6.0, 6.9, and 7.6 Hz for AMP, 8-Cl-AMP, and 8-amino-AMP, respectively (29). The 3JH-1',H-2' value obtained for AMP was in agreement with published values for ATP (6.0 Hz) (35) and 8-amino-AMP (7.9 Hz) (36). These data show that C-8 substitution with chlorine increased the relative population of the C-2'-endo sugar pucker from 60% (AMP) to 69% for 8-Cl-AMP and that substitution with an amine group further shifted the equilibrium to 76% of the C-2'-endo conformer for 8-amino-AMP. These results suggest that the relative proportion of the C-2'-endo form increases with the relative size of the C-8 group; the van der Waals radius of a hydrogen atom is significantly less than that of chlorine or an amino group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Modification at C-8 of ATP disrupts normal polyadenylation of RNA by yPAP, and the modified nucleotide derivatives inhibit poly(A) tail synthesis via a range of mechanisms. Several factors appear to be involved with yPAP substrate selection of triphosphate analogs, including the chemical nature and site of modification. Our results suggest that substitution at C-2 of the aromatic heterocycle does not significantly affect polyadenylation efficiency in the absence of ATP. Both C-2-halogenated and amine-modified ATP derivatives showed chain extension beyond a single nucleotide insertion. In contrast, the C-8-modified analogs examined in this study were either chain terminators or inhibitors, depending on the type of modification. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP were all chain terminators like 3'-dATP, indicating that this class of analogs halts polyadenylation by direct incorporation into nascent poly(A) tails. Interestingly, 8-Br-ATP and 8-Cl-ATP are poor substrates for yPAP. The substrate specificity for yPAP is fairly broad; several dNTPs and ddNTPs can be used to extend primers with yPAP (25). Introduction of a halogen group at C-8 completely eliminated the substrate properties of 8-Br-ATP and 8-Cl-ATP for yPAP. However, incorporation of 8-Cl-AMP at the 3'-terminal site of an RNA primer blocked further polyadenylation by yPAP.

ATP analogs that were modified at C-2 and C-8 were also examined as inhibitors of yPAP in the presence of ATP, and they displayed inhibition profiles that paralleled the observed substrate specificities of each analog. Substitution at C-2 of ATP did not appear to strongly affect the ability of yPAP to synthesize poly(A) tails with ATP. Neither 2-Cl-ATP nor 2-amino-ATP had a marked effect on chain extension with ATP. Nitrogenous modification at C-8 resulted in a moderate decrease in poly(A) tail length at higher concentrations, as observed with 8-amino-ATP, 8-azido-ATP, and 8-aza-ATP. However, halogen modification at C-8 had a major effect on polyadenylation and resulted in a significant reduction in poly(A) tail length with an increasing concentration of 8-halogenated ATP in the presence of ATP. 8-Cl-ATP was also the most potent polyadenylation inhibitor of the analogs examined.

The results obtained from the polyadenylation assays indicate that several factors are involved in yPAP substrate selection and primer extension. Polyadenylation activity is sensitive to modification at C-8 of the adenine heterocycle, and distinct trends were observed among classes of types of modifications. The C-8-halogenated derivatives, 8-Br-ATP and 8-Cl-ATP, were the poorest substrates and the most potent inhibitors of polyadenylation. The low substrate efficiency and inhibitory properties of 8-Br-ATP and 8-Cl-ATP with yPAP may arise from several mechanisms. First, yPAP exhibits burst kinetics, which is associated with activation of the nucleotide substrate by formation of an enzyme-AMP intermediate, followed by nucleotidyl transfer to the RNA primer (37). A related polymerase, vaccinia virus PAP, has been shown to form a covalent nucleotidyl-enzyme complex with 3'-dATP (38). Halogen modification at C-8 may negatively affect the ability of the enzyme to activate the ATP substrate or to transfer the AMP group from the enzyme to the RNA substrate. Alternatively, because both bromine and chlorine substituents are relatively good leaving groups, irreversible alkylation of yPAP by 8-Br-ATP or 8-Cl-ATP may inactivate the enzyme and produce the observed inhibition profile. Further studies will be required to assess the predominant mechanism for yPAP inhibition by the C-8-halogenated ATP analogs. However, mechanisms based on covalent enzyme inactivation do not address the more substitution-resistant analogs such as 8-amino-ATP, 8-azido-ATP, and 8-azaATP. An alternative hypothesis is that C-8 modification may induce glycosidic bond sugar pucker conformational changes and confer inhibitory properties to the ATP analog.

Although a number of C-8-modified adenosine analogs have been shown to adopt a syn-glycosidic conformation (39–41), our experiments indicate that AMP and 8-Cl-AMP display similar distributions of syn- and anti-glycosidic conformation in aqueous solution. In contrast, 8-amino-AMP appears to adopt a predominantly anti conformation, consistent with previous literature reports (36). For comparison, other ATP-derived polyadenylation inhibitors, Ara-ATP and 2'-dATP, both impair polyadenylation efficiency and should have similar base conformations as the natural polyadenylation substrate, ATP. Therefore, the glycosidic base conformation may not be the determining factor in substrate recognition by yPAP. Our studies suggest that sugar pucker conformation of the 3'-terminal nucleotide of the RNA primer may be an important factor for substrate selection with yPAP. Earlier work demonstrated that the identity of the 3'-terminal nucleotide of the RNA primer is a critical determinant of polyadenylation efficiency (42). Incorporation of two to three 2'-dAMP residues at the 3' terminus of RNA substrates significantly decreases polyadenylation efficiency. Conversely, the addition of two to three AMP residues to the 3' terminus of DNA primers, which normally are not substrates for polyadenylation, results in poly(A) tail addition by yPAP. These results suggest that the 3'-RNA-binding site of yPAP strongly discriminates against deoxyribonucleotidyl residues at the 3'-end of RNA ribonucleotides (42). The main conformational difference between ribonucleotides and deoxyribonucleotides lies in the sugar pucker conformation: the preferred sugar conformation is C-3'-endo for ribonucleotides and C-2'-endo for deoxyribonucleotides (43). Similarly, arabinonucleotides have been shown to adopt predominantly DNA-like conformations and favor C-2'-endo sugar pucker (44). C-8 modification of ATP may alter the sugar pucker equilibrium to increase the relative population of the C-2'-endo form over the natural C-3'-endo form. The resulting incorporation of modified ATP analogs into 3'-terminal positions may alter the terminal ribose pucker conformation, such that the 3'-hydroxyl group is positioned unfavorably for primer extension. This hypothesis is consistent with the observed chain termination by 8-amino-ATP, 8-azido-ATP, and 8-aza-ATP and the inability of yPAP to extend primers containing 3'-terminal 8-Cl-AMP sites. Our NMR spectroscopic results support the hypothesis that C-8 substitution influences sugar pucker conformation, which may affect yPAP efficiency. As a result, incorporation of 8-modified adenosine analogs into RNA may have significant consequences; naturally occurring cleaved mammalian mRNA frequently is terminated by a CA dinucleotide at the 3'-end (8). Although 8-Cl-ATP does not serve as a direct substrate for yPAP, 8-Cl-AMP insertion may occur during transcription with RNA polymerase II (10). Thus, incorporation of 8-modified AMP nucleotides at sites critical for the initiation of polyadenylation may inhibit pre-mRNA cleavage and polyadenylation in cellular systems.

We have demonstrated that C-8-modified adenosine derivatives inhibit poly(A) tail synthesis by yPAP. Polyadenylation inhibition represents one mode of action for 8-Cl-Ado and 8-amino-Ado in hematological malignancies, among several possible mechanisms. RNA-directed drugs may offer a valuable strategy in targeting indolent cancers, such as multiple myeloma, which do not actively synthesize DNA and thus are not amenable to traditional DNA-directed therapies. The specificity of C-8-modified analogs toward inhibition of RNA processes may be instrumental in the development of new analogs. Further studies using human nuclear extracts will be conducted to elucidate the effects of 8-modified adenosine analogs on polyadenylation events in human cells.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant RO1 CA85915-02 and Leukemia and Lymphoma Society Grant 6505-00. The MALDI-TOF mass spectrometer was purchased with funds provided by National Institutes of Health Scientific Instrumentation Grant 1-S10-RR13810. The Center for Structural Biology at Northwestern University was supported by the Robert H. Lurie Comprehensive Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S3 and Table S1. Back

{ddagger} To whom correspondence should be addressed: Dept. of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208-3113. Tel.: 847-467-7636; Fax: 847-491-7713; E-mail: t-sheppard{at}northwestern.edu.

1 The abbreviations used are: Cl, chloro; Ara-ATP, 9-{beta}-D-arabinofuranosyladenine 5'-triphosphate; yPAP, yeast poly(A) polymerase; RP-HPLC, reversed-phase high pressure liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Br, bromo; ESI-MS, electrospray ionization mass spectrometry; TEMED, N,N,N',N''-tetramethylethylenediamine; dPAGE, denaturing polyacrylamide gel electrophoresis; BSA, bovine serum albumin; DQF-COSY, double quantum-filtered correlation spectroscopy. Back

2 N. L. Krett, K. M. Davies, M. Ayres, C. Ma, C. Nabhan, V. Gandhi, and S. T. Rosen, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Professor Ishwar Radhakrishnan and Dr. Ben Ramirez (Northwestern University) for assistance with NMR spectroscopic studies. We acknowledge use of instruments in the Keck Biophysics Facility and the Analytical Services Laboratory of Northwestern University.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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