Báo cáo khoa học: Mouse RS21-C6 is a mammalian 2¢-deoxycytidine 5¢-triphosphate pyrophosphohydrolase that prefers 5-iodocytosine pdf

Mouse RS21-C6 is a mammalian 2¢-deoxycytidine 5¢-triphosphate pyrophosphohydrolase that prefers 5-iodocytosine Mari Nonaka, Daisuke Tsuchimoto, Kunihiko Sakumi and Yusaku Nakabeppu Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Keywords 5-I-dCTP; CpG methylation; dCTPase; modified nucleotide; nucleotide metabolism Correspondence D Tsuchimoto, Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan Fax: +81 92 642 6804 Tel: +81 92 642 6802 E-mail: daisuke@bioreg.kyushu-u.ac.jp (Received September 2008, revised January 2009, accepted 12 January 2009) doi:10.1111/j.1742-4658.2009.06898.x Free nucleotides in living cells play important roles in a variety of biological reactions, and often undergo chemical modifications of their base moieties As modified nucleotides may have deleterious effects on cells, they must be eliminated from intracellular nucleotide pools We have performed a screen for ITP-binding proteins because ITP is a deaminated product of ATP, the most abundant nucleotide, and identified RS21-C6 protein, which bound not only ITP but also ATP Purified, recombinant RS21-C6 hydrolyzed several canonical nucleoside triphosphates to the corresponding nucleoside monophosphates The pyrophosphohydrolase activity of RS21C6 showed a preference for deoxynucleoside triphosphates and cytosine bases The kcat ⁄ Km (s)1Ỉm)1) values were 3.11 · 104, 4.49 · 103 and 1.87 · 103 for dCTP, dATP and dTTP, respectively, and RS21-C6 did not hydrolyze dGTP Of the base-modified nucleotides analyzed, 5-I-dCTP showed an eightfold higher kcat ⁄ Km value compared with that of its corresponding unmodified nucleotide, dCTP RS21-C6 is expressed in both proliferating and non-proliferating cells, and is localized to the cytoplasm These results show that RS21-C6 produces dCMP, an upstream precursor for the de novo synthesis of dTTP, by hydrolyzing canonical dCTP Moreover, RS21-C6 may also prevent inappropriate DNA methylation, DNA replication blocking or mutagenesis by hydrolyzing modified dCTP In living organisms, nucleotides play various roles, as signal transmitters, molecular switches, coenzymes or as carriers of energy, in addition to their important role as precursors of DNA ⁄ RNA synthesis For example, ATP is a major carrier of energy, a phosphate group donor in kinase reactions, and an extracellular signal transmitter GTP is a molecular switch in signal transduction pathways and an initiator complex for translation UTP and CTP are utilized to form active intermediates in the biosynthesis of polysaccharides or phospholipids, respectively In such roles, recognition of nucleotides by specific proteins is very important Intracellular nucleotides, however, undergo chemical modifications caused by endogenous reactive molecules, such as reactive oxygen species, or by exogenous factors, such as chemicals and ionizing irradiation Chemical modification may alter the characteristics of nucleotides, including their recognition by proteins Abbreviations 2-Cl-dATP, 2-chloro-(2¢-deoxy)adenosine 5¢-triphosphate; 2-OH-(d)ATP, 2-hydroxy-(2¢-deoxy)adenosine 5¢-triphosphate; 5-Br-dCTP, 5-bromo-2¢deoxycytidine 5¢-triphosphate; 5-F-dUTP, 5-fluoro-2¢-deoxycytidine 5¢-triphosphate; 5-I-dCTP, 5-iodo-2¢-deoxycytidine 5¢-triphosphate; 5-Me-dCTP, 5-methyl-2¢-deoxycytidine 5¢-triphosphate; 5-OH-dCTP, 5-hydroxy-2¢-deoxycytidine 5¢-triphosphate; 8-oxo-(d)GTP, 8-oxo-(2¢deoxy)guanosine 5¢-triphosphate; DCTD, dCMP deaminase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NTP, nucleoside 5¢-triphosphate; RNR, ribonucleotide reductase; TS, thymidylate synthase 1654 FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS M Nonaka et al Some modified deoxynucleotides are incorporated into DNA by DNA polymerases and accumulate in newly synthesized DNA This may prevent DNA replication or transcription, resulting in cell death and degenerative diseases in humans [1] Normal functions of nucleotides, other than DNA synthesis, may also be adversely affected by modified nucleotides Cells are equipped with defense systems against such modified nucleotides Some modified nucleotides in intracellular nucleotide pools are hydrolyzed by specific enzymes [1,2] Of these enzymes, dUTPase and MTH1 are the best studied in human cells The former hydrolyzes deoxyuridine triphosphate to prevent its incorporation into DNA The latter hydrolyzes oxidized purine nucleoside triphosphates, including 8-oxo-(deoxy)guanosine triphosphate [8-oxo-(d)GTP] and 2-hydroxy(deoxy)adenosine triphosphate [2-OH-(d)ATP], to the corresponding (deoxy)nucleoside monophosphates and pyrophosphates to avoid their incorporation into DNA or RNA [3] The spontaneous mutation rate in MTH1-null mouse embryonic stem cells was twofold higher than that in wild-type cells Further, MTH1null mice showed more frequent tumorigenesis in the liver compared to wild-type mice [4] In addition to oxidization, deamination of the amino group is another major chemical modification that occurs in purine nucleotides Deamination of the amino group at C6 of adenine or C2 of guanine generates hypoxanthine or xanthine, respectively Thus, (d)ITP and (d)XTP are generated from (d)ATP and (d)GTP, respectively Incorporation of these modified nucleotides into DNA during DNA replication or into RNA during transcription results in gene mutations or the synthesis of abnormal proteins because hypoxanthine and xanthine can mis-pair with cytosine or thymine, respectively Recently, mammalian inosine triphosphate pyrophosphohydrolases (ITPases) have been reported to hydrolyze deaminated purine nucleoside triphosphates to the corresponding nucleoside monophosphates and pyrophosphates [5,6] ITPasenull mice, in which accumulation of ITP was observed, showed abnormal development and died within 14 days after birth (M Behmanesh, K Sakumi, S Toyokuni, S Oka, Y Ohnishi, D Tsuchimoto & Y Nakabeppu, unpublished results) The ITP that accumulates in these mice may have deleterious effects on cell functions, for example via DNA ⁄ RNA synthesis, or, because of its structural similarity to ATP, by interaction with ATP-related proteins In the present study, we prepared ITP-agarose and purified ITP-binding proteins to identify additional ITP-hydrolyzing enzymes or target proteins whose function can be inhibited by ITP As a result, we iden- A mammalian dCTPase that prefers 5-iodocytosine tified RS21-C6, which was previously reported to be a thymocyte development-related molecule [7], as well as ITPase, as ITP-binding proteins Because RS21-C6 contains a typical MazG domain conserved in the bacterial NTP pyrophosphatase MazG, it has been described as a member of the all-a NTP pyrophosphohydrolase superfamily with all-a helix structures [8] A preliminary structure of RS21-C6 without substrate has been initially determined [9] Recently, it was shown that RS21-C6 hydrolyzes 5-methyl-dCTP, and the crystal structure of truncated RS21-C6 complexed with 5-methyl-dCTP indicated that tetramer formation is required for substrate binding [10] We examined the NTP pyrophosphohydrolase activity of purified recombinant RS21-C6 protein towards various nucleotides, and found that it hydrolyzes some deoxynucleotides, particularly dCTP, but not dITP or ITP Furthermore, we found that iodination at C5 of cytosine significantly increases the kcat ⁄ Km value of RS21-C6 Results Preparation of ITP-agarose We prepared ITP-agarose from ATP-agarose as described in Experimental procedures Analysis of bases excised from agarose beads revealed that most adenine bases on the agarose were converted to hypoxanthine after deamination (Fig 1) We also confirmed that most free ATP was converted to ITP after the same treatment (data not shown), demonstrating that the nucleotides on the treated agarose were ITP Quantification of released bases indicated that the amounts of nucleotide on ATP- and ITP-agarose were 17.3 and 2.6 nmol per 25 lL bed volume, respectively ITP-binding proteins ITP-binding proteins were purified from mouse thymocyte extract by a pulldown method using ITP-agarose Proteins were then fractionated by SDS–PAGE (Fig 2A) After staining the acrylamide gel, we chose ten ITP-specific bands, and, from these, identified 11 proteins by LC-MS ⁄ MS analysis (Table 1) We detected ten peptides of ITPase and four peptides of RS21-C6 in bands and 5, respectively Specific binding of ITPase to ITP-agarose was confirmed by western blot analysis of pulldown samples using antiITPase serum [5] (Fig 2B, upper panel) To analyze the interaction of RS21-C6 with ITP in detail, RS21-C6 cDNA and recombinant RS21-C6 protein were prepared as described in Experimental procedures We performed pulldown experiments, using ITP-agarose FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS 1655 A mammalian dCTPase that prefers 5-iodocytosine 248.5 nm (AU) 0.06 M Nonaka et al Table ITP-binding proteins identified by LC-MS ⁄ MS analysis Before deamination Band no 0.04 0.02 248.5 nm (AU) 0.06 Acetyl coenzyme A acetyltransferase precursor Glyceraldehyde-3-phosphate dehydrogenase Glutathione S-transferase Inosine triphosphatase Similar to ISOC2 protein RS21-C6 Phospholipid hydroperoxide glutatione peroxidase Ribosomal protein L30 Divalent cation tolerant protein CUTA isoform Unidentified Isochorismatase domain-containing Es protein Unidentified 0.00 12 16 20 After deamination 0.04 0.02 * 0.00 12 Retention time (min) 16 20 10 114 84.7 gi|21450129 gi|50233866 gi|2781337 gi|31982664 gi|20818892 gi|13435502 gi|2522259 gi|6677783 gi|62198210 gi|31541909 gi|20070420 from cell extracts of Escherichia coli BL21-CodonPlus (DE3)-RIL that had been transformed with pET3a: RS21-C6 and induced for RS21-C6 expression Recombinant RS21-C6 protein in bacterial cell extracts also bound to both ITP- and ATP-agarose (Fig 2C) D Agarose ATP-agarose (kDa) 25 (kDa) ITP-agarose B ITP-agaros e A ATP-agaros e Fig Preparation of ITP-agarose ATP-agarose were incubated in M HCl before (upper graph) or after (lower graph) deamination, and the bases released were analyzed by HPLC The peaks indicated by an open arrowhead and a closed arrowhead were compared with peaks of standard samples, and were identified as adenine base and hypoxanthine base, respectively The peak indicated by an asterisk was also observed in a sample released from agarose without any nucleotide, suggesting that it was derived from the carrier agarose (data not shown) NCBInr accession no Protein description (kDa) 250 150 100 75 ITPase 17 25.7 50 37 47.3 (kDa) 25 20 10 15 Recombinant RS21-C6 15 Agarose 20 C ITP-agarose 25.7 17.4 25 ATP-agarose 31.6 RS21-C6 17.4 Recombinant RS21-C6 Fig Purification of ITP-binding proteins (A) Pulldown of ITP-specific proteins from mouse thymocyte extract Proteins were pulled down using ATP- or ITP-agarose and then separated by SDS–PAGE The lanes were loaded with samples pulled down from 5.0 · 107 cells The gel was subjected to silver staining Arrowheads indicate the numbered ITP-specific bands that were recovered and subjected to a mass spectrometry (B) Western blot of pulled down samples with anti-ITPase serum and anti-RS21-C6 Ig Samples pulled down from 2.5 · 107 cells were loaded on the gel as described in (A) (C) Pulldown of recombinant RS21-C6 Recombinant RS21-C6 protein was expressed in E coli Binding proteins were pulled down using agarose beads, separated by SDS–PAGE and stained by silver staining (D) Purification of recombinant RS21-C6 protein Recombinant RS21-C6 protein, expressed in E coli, was purified as described in Experimental procedures The purified protein (100 ng) was separated by SDS–PAGE and stained by silver staining 1656 FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS M Nonaka et al A mammalian dCTPase that prefers 5-iodocytosine Expression of endogenous RS21-C6 A pcDNA :RS21-C6 pcDNA BALB/3T3 NIH/3T3 WEHI231 J774A.1 3BW5147.3 Thymocyte (kDa) 20 (kDa) 114 84.7 RS21-C6 15 47.3 GAPDH 31.3 25.7 C W N Mt Cyt RS21-C6 17.4 RS21-C6 Lamin B HSP60 GAPDH GAPDH 20 15 10 reb rum reb ellu m Ey Sp e ina l co rd He art Kid ne y Liv er Es Lung op gu Ly s mp hn od e Bo Sple en ne ma rro w Th ym us Te stis Ute r Sto us ma Sa liva ch ry gla nd Sk Ov ele ary tal mu Th scl yro e id gla nd Ce Relative expression D Ce Fig Expression of endogenous RS21-C6 (A) Western blot analysis of RS21-C6 protein in various mouse cell lines A whole-cell extract from 1.0 · 105 cells of each cell line was loaded in each lane Proteins were separated by SDS–PAGE, and then transferred to a poly(vinylidene difluoride) membrane Signals for RS21-C6 protein and of GAPDH were detected using anti-RS21-C6 and antiGAPDH Ig, respectively (B) Western blot analysis of endogenous or recombinant RS21-C6 proteins in A20 cells A20 cells were transfected with plasmids expressing recombinant RS21-C6 or with control vectors by electroporation The cells were incubated for 24 h, and whole-cell extracts from 1.0 · 105 cells were loaded on each lane (C) Intracellular localization of RS21-C6 protein Aliquots (20 lg protein) from whole-cell extract (W) or each cell fraction were loaded into each lane Lamin B, HSP60 or GAPDH were detected as nuclear (N), mitochondrial (Mt) or cytoplasmic (Cyt) markers, respectively (D) Real-time quantitative PCR analysis of RS21-C6 expression in various mouse tissues The mRNA expression levels of RS21-C6 were normalized to those of 18S rRNA Error bars represent SD (n = 3) The expression level of RS21-C6 in spleen was arbitrarily set as 1.0, and the expression levels in the other tissues are expressed relative to that in spleen A20 B pIRES2-EGFP :RS21-C6 Western blot analysis using anti-RS21-C6 Ig and whole-cell extracts from several mouse cell lines produced an intense band corresponding to a polypeptide with a molecular mass of about 19 kDa in each lane (Fig 3A), although additional, non-specific bands were detected in some lanes We then transfected the mouse B cell lymphoma line A20 individually with two plasmids to express non-tagged recombinant RS21-C6, and independently transfected vector controls without inserts After incubation for 24 h, whole-cell extracts were prepared and subjected to western blot analysis using anti-RS21-C6 We detected a band with a very intense signal that corresponded to a size of approximately 19 kDa in each of the samples overexpressing RS21-C6 We detected a band with identical mobility but a weak signal in each of the vector control samples, indicating that anti-RS21-C6 specifically reacts pIRES2-EGFP TrxA-RS21-C6 protein was expressed from pET32a(+):RS21-C6 and was used as an antigen to prepare anti-RS21-C6 rabbit serum Western blot analysis, using affinity-purified anti-RS21-C6 Ig, showed that endogenous RS21-C6 also binds to both ITP- and ATP-agarose (Fig 2B, lower panel) Glyceraldehyde3-phosphate dehydrogenase (GAPDH), detected in band 2, bound to the negative control deaminated agarose, as well as to ITP-agarose, as shown by western blot analysis with anti-GAPDH Ig, suggesting that the binding of GAPDH was not nucleotide-specific (data not shown) The non-tagged recombinant RS21-C6 protein was purified to a nearly homogeneous state by SDS– PAGE to analyze its enzyme activity (Fig 2D) Its molecular mass was estimated, based on its SDS– PAGE mobility, as approximately 19 kDa, which is almost identical to the calculated molecular weight of 18 783 FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS 1657 A mammalian dCTPase that prefers 5-iodocytosine A 0.08 M Nonaka et al with both recombinant RS21-C6 and mouse RS21-C6 that is endogenous to A20 cells (Fig 3B) We then analyzed the intracellular localization of the RS21-C6 protein Nuclear, mitochondrial and cytosolic fractions were prepared from mouse liver Western blot analysis of each fraction revealed that RS21-C6 is exclusively located in the cytosol (Fig 3C) Finally, we examined the expression levels of RS21-C6 mRNA by real-time quantitative PCR, and found that RS21-C6 is ubiquitously expressed and that expression was highest in the liver and heart, and to a lesser extent the salivary gland (Fig 3D) dCTP + buffer 0.04 272 nm (AU) 0.00 0.08 dCTP + RS21-C6 0.04 0.00 0.08 dCMP standard 0.04 Nucleoside triphosphate pyrophosphohydrolase activity of RS21-C6 protein 0.00 80 60 40 20 10 20 30 40 Temperature (°C) 50 60 PIPES-Na Tris-HCl AMPD-HCl 80 60 40 20 pH 10 Mg2+ Mn2+ 100 Product (%) D 100 Product (%) C 50 100 Product (%) B 40 10 20 30 Retention time (min) 80 60 40 20 0 20 40 60 80 Metal ion concentration (mM) 100 Product (%) E 100 80 60 40 NaCl KCl 20 1658 In a preliminary analysis, using canonical nucleotides, purified RS21-C6 protein showed strong pyrophosphohydrolase activity on dCTP, producing dCMP (Fig 4A, middle panel) We also analyzed the reaction product using BIOMOL GREEN reagent (Enzo Biochem, Inc., New York, NY, USA) and detected no free phosphate, indicating that RS21-C6 hydrolyzes dCTP to dCMP and pyrophosphate (data not shown) Next, we analyzed the optimal conditions for the pyrophosphohydrolase activity of RS21-C6 using dCTP as a substrate RS21-C6 showed a temperature-dependent increase of activity up to 60 °C (Fig 4B) RS21-C6 demonstrated strongest activity at pH 9.5, the highest pH analyzed here (Fig 4C) The divalent metal cation requirements of RS21-C6 were tested using MgCl2 and MnCl2 No activity was detected in reactions without added metals, and maximum activity was measured in reactions containing 100 mm MgCl2 At 100 mm, MnCl2 did not support full activity (Fig 4D) RS21-C6 showed the same activity with various concentrations of KCl between and 1000 mm (Fig 4E) NaCl moderately reduced RS21-C6 activity Based on these results and in view of physiological conditions, we 200 400 600 800 Salt concentration (mM) 1000 Fig dCTP pyrophosphohydrolase activity of RS21-C6 protein (A) Hydrolysis of dCTP to dCMP by RS21-C6 Substrate dCTP (300 lM) was incubated for 20 in reaction buffer supplemented with 50 nM of purified RS21-C6 protein Reaction products were analyzed by HPLC (middle panel), and were compared with substrate dCTP incubated in the reaction buffer without RS21-C6 (upper panel), and with standard dCMP prepared in the reaction buffer (lower panel) The dependency of RS21-C6 activity on temperature (B), buffer pH (C), divalent metal cations (D) and salts (E) was analyzed The amounts of dCMP produced were measured and are shown as percentages of the highest value Each point and error bar indicates the mean and standard deviation of three reactions FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS M Nonaka et al A mammalian dCTPase that prefers 5-iodocytosine Table Kinetic parameters for nucleoside triphosphate pyrophosphohydrolase activity of RS21-C6 protein Km (lM) ATP GTP CTP UTP ITP 2-OH-ATP dATP dGTP dCTP dTTP dUTP dITP 2-OH-dATP 2-Cl-dATP 8-oxo-dGTP 5-OH-dCTP 5-Me-dCTP 5-Br-dCTP 5-I-dCTP 5-Cl-dCTP 5-F-dUTP dCDP dADP No activity No activity 529 No activity No activity No activity 118 No activity 44.1 407 484 No activity 169 107 No activity 164 48.5 21.7 3.9 50.0 304 No activity No activity kcat (s)1) kcat ⁄ Km (s)1ỈM)1) 0.31 156 0.53 4490 1.37 0.76 0.76 31 100 1870 1570 1.39 1.57 8220 14 700 0.87 1.33 1.49 0.94 1.50 0.58 5300 27 400 68 700 241 000 30 000 1900 performed further analysis of RS21-C6 activity under the conditions described in Experimental procedures The substrate concentration was set at 10, 30, 100 or 1000 lm, except for that of 5-I-dCTP, which was set at A µM 3.8 µM 7.8 µM 15.6 µM 31.3 µM [Product] (µM) 10 Time (min) 5 10 Time (min) 15 20 C rate)–1 (s·µM–1) 50 (Reaction Reaction rate (µM·min–1) Fig Kinetic analysis of the reaction of RS21-C6 with 5-I-dCTP The concentration of 5-I-dCTP substrate was set at 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 or 1000 lM (A) Enzyme reaction curves (B) Michaelis– Menten plot using the results at substrate concentrations from to 62.5 lM, showing the saturation pattern (C) A Lineweaver– Burk plot prepared using all the results 30 0 B 62.5 µM 125 µM 250 µM 500 µM 1000 µM 60 [Product] (µM) Substrate 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 or 1000 lm, because the Km value for 5-I-dCTP is < 10 lm The Km and kcat values at 50 nm RS21-C6 for various nucleotides were determined from Lineweaver–Burk plots, and are shown in Table Except for CTP, RS21-C6 did not hydrolyze the analyzed ribonucleotides, even ITP For the analyzed deoxynucleotides, the kcat ⁄ Km values for dCTP, dATP and dTTP were 3.11 · 104, 4.49 · 103 and 1.87 · 103, and RS21-C6 did not hydrolyze dGTP and dITP Thus, RS21-C6 shows a preference for cytosine base and deoxyribose Among the base-modified nucleotides, 5-I-dCTP had an eightfold higher kcat ⁄ Km value compared to its corresponding unmodified deoxynucleotide, dCTP We analyzed the hydrolysis of dCTP by RS21-C6 in the presence of ITP, and found that ITP did not inhibit it even at 500 lm (data not shown) Enzyme reaction curves, a Michaelis–Menten plot and a Lineweaver–Burk plot for 5-I-dCTP are shown in Fig The oligomeric structure of RS21-C6, together with the conformational flexibility of its active sites, suggests cooperativity between the sites and allosteric regulation However, we obtained a typical Michaelis–Menten-type saturation curve rather than the sigmoid curve typical of non-Michaelis–Mententype reactions This result indicates that the RS21-C6 tetramer does not show any cooporative binding of substrates to the multiple active sites under the present conditions 25 0 10 20 30 40 50 [Substrate] (µM) FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS 60 70 –0.2 0.2 [Substrate]–1 (µM–1) 0.4 1659 A mammalian dCTPase that prefers 5-iodocytosine M Nonaka et al Discussion We have identified ITPase, a known ITP-hydrolyzing enzyme, as an ITP-binding protein, using ITP-agarose This indicates that our screening technology is a valid approach for identification of novel proteins that bind various modified nucleotides RS21-C6, another protein identified in this study, was an ITP-binding protein which can also bind ATP Because of its homology in amino acid sequence to known nucleoside triphosphate (NTP) pyrophosphohydrolases, including bacterial MazG proteins [8], we analyzed its catalytic activity for hydrolyzing canonical nucleotides We showed that RS21-C6 is a deoxynucleoside triphosphate pyrophosphohydrolase that prefers dCTP In mammalian cells, no other protein has been reported as a pyrimidine dNTP-specific pyrophosphohydrolase, although Orf135 and iMazG, a novel bacterial MazG protein, have been reported as bacterial dCTPases [11,12] The likely biological unit of RS21-C6 is a tetramer [10], suggesting that the multivalency of the RS21-C6 tetramer may stabilize its interaction with ITP immobilized on agarose beads This may explain why RS21-C6, which is not an ITP-specific protein, has been identified as an ITP-binding protein It has been shown that the substrate-binding pocket of RS21-C6 comprises several residues, including His38, Trp47, Trp73, Tyr102, Glu63, Glu66, Glu95 and Asn98, that the nitrogenous base and deoxyribose of 5-methyl-dCTP are located in a hydrophobic cavity, and that the phosphate groups interact with the four electronegative amino acid residues [10] Among the known all-a-NTP pyrophosphohydrolases, we found that residue Asn125 in RS21-C6 is conserved in various dUTPases [Campylobacter jejuni dUTPase (CjdUTPase), Leishmania major dUTPase, Trypanosoma cruzi dUTPase (TcdUTPase)], dCTPases (enterobacteria phage T2 dCTPase, enterobacteria phage T4 dCTPase, bacteriophage RB15 dCTPase), and iMazG [8,12] Moroz et al and Harkiolaki et al analyzed the crystal structures of CjdUTPase [13] and TcdUTPase [14], respectively, with their substrates They showed that Asn179 of CjdUTPase and Asn201 of TcdUTPase, the residues corresponding to Asn125 of RS21C6, bind to the 2¢-deoxyribose moiety of substrates This residue is not conserved in either the HisE family, which has phosphoribosyl-ATP pyrophosphatase activity (E coli HisIE, Corynebacterium glutamicam HisE, Pyrococcus furiosus HisIE, Saccharomyces cerevisiae HIS4, Arabidopsis thaliana HisIE), or the MazG family (SSO12199, E coli MazG, Thermotoga maritime MazG, Bacillus subtilis YABN, Streptomyces cacaoi YBL1) Because enzymes in the HisE and MazG fami1660 lies but not those of the dUTPase, dCTPase, iMazG and RS21-C6 families hydrolyze ribonucleotides, we suggest that the Asn125 residue in RS21-C6 may be involved in the preference for deoxyribose sugar Wu et al [10] did not mention an interaction between Asn125 and the substrate 5-methyl-dCTP in their analysis of the crystal structure of RS21-C6 with the substrate They used the core fragment of RS21-C6 (RSCUT: residues 21–126) in which the Asn125 residue is located very close to the C-terminal end Therefore, Asn125 might not be appropriately located in the truncated molecule Recognition of the cytosine base by RS21-C6 appears to be supported by the His38 residue in helix 1, which forms a hydrogen bond with the O2 of the cytosine base [10] This corresponds to the glutamine residues in the first helix of dUTPases (Gln14 of CjdUTPase or Gln22 of TcdUTPase), which form a hydrogen bond with the O2 of a uracil base [8] It has been shown that His58 of CjdUTPase [13] and Trp61 of TcdUTPase [14] form a hydrogen bond with O4 of the uracil base, and this appears to be involved in their discrimination of uracil from cytosine These residues are not conserved in RS21-C6, supporting its lower affinity for dUTP in comparison to dCTP as revealed in the present study RS21-C6 has essentially similar affinities towards both dCTP and 5-methyl-dCTP, indicating that there may not be a specific residue that recognizes the methyl group at the C5 position However, halogenation at the C5 position, particularly iodination, significantly increased its affinity to RS21-C6 It is possible that one of the residues that form the substrate-binding pocket may recognize the iodine at the C5 position An analysis of the crystal structures of RS21-C6 complexed with 5-I-dCTP will help to delineate the structural basis for its substrate recognition What is the biological role of dCTPase in mammalian cells? RS21-C6 hydrolyzes dCTP and produces dCMP In mammalian cells, deamination of dCMP by dCMP deaminase is the most important pathway for dUMP production dUMP is then converted to dTMP by thymidylate synthase [15–18], and dTMP is converted to dTTP by a two-step phosphorylation reaction The activity of dCMP deaminase is positively regulated by dCTP, and negatively by dTTP [19] Hence, the conversion of dCTP to dTTP appears to be a key pathway for regulating the ratio of dCTP ⁄ dTTP in the nucleotide pool Based on pulse-chase experiments using [5-3H]cytidine, Bianchi et al [20] estimated that dCTP was incorporated into DNA at a rate of 16 pmolỈmin)1 in rapidly growing mouse 3T6 cells, and that dTMP was formed from free dCMP at a rate FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS M Nonaka et al of 10 pmolỈmin)1 A defect in dCMP deaminase in hamster fibroblasts was reported to cause an imbalanced dCTP ⁄ dTTP ratio, and to mildly affect the fidelity of DNA replication [21] dCTP is not an effector molecule that allosterically controls ribonucleotide reductase (RNR) Therefore, cells must have other mechanisms to regulate the cytosolic dCTP concentration In this regard, dCTP pyrophosphohydrolase plays an important role in avoiding accumulation of excess dCTP and supplying sufficient levels of dCMP, an upstream precursor of de novo synthesis of dTTP Expression of RS21-C6 mRNA was detected in all tissues examined in this study, and was particularly high in the liver and heart These data suggest that RS21-C6 plays a role in both proliferating and non-proliferating cells Additionally, we found that RS21-C6 was localized to the cytosol In mammalian cells, two types of RNR, R1 ⁄ R2 RNR and R1 ⁄ p53R2 RNR, regulate the synthesis of dNTPs for DNA replication [22,23] In proliferating cells, the R1 ⁄ R2 RNR complex, which consists of R1 and R2 subunits, is localized to the cytoplasm and supplies deoxynucleotides for nuclear DNA synthesis [22] Even in non-proliferating cells, dNTPs are necessary for mitochondria DNA replication These cells express the p53R2 subunit instead of the R2 subunit in the cytoplasm Mitochondrial DNA depletion caused by mutations in the RRM2B gene, which encodes the p53R2 subunit, demonstrates that the R1 ⁄ p53R2 RNR complex plays a critical role in dNTP supply for mitochondrial DNA replication [24] Similarly to the RS21-C6 gene, expression of the RRM2B gene is found in many tissues, in contrast to the R2 subunit, which is undetectable in the heart, brain and muscle [25] dTMP and thymidine synthesized in the cytoplasm are imported into the mitochondria, phosphorylated by mitochondrial enzymes and used for mitochondrial DNA replication [26] On the other hand, it has been reported that dCTP transport activity exists in human mitochondria [27] These reports raise the possibility that the cytosolic concentration of dCTP and dTMP influences the balance of mitochondrial deoxypyrimidine nucleotide pools (Fig 6A) Several members of the NTP pyrophosphohydrolase family have also been shown to eliminate non-canonical nucleotides from the intracellular NTP pool Here, we have shown that RS21-C6 has the highest kcat ⁄ Km value for the modified deoxynucleotide 5-IdCTP of the various nucleotides examined, including its corresponding canonical deoxynucleotide, dCTP Our data indicate that 5-I-dCTP or its analogs might be true substrates of RS21-C6 It is unlikely that the intracellular level of ITP prevents RS21-C6 activity, A mammalian dCTPase that prefers 5-iodocytosine A B Fig Models of two biological roles of RS21-C6 protein (A) RS21-C6 may supply dCMP as an upstream precursor of de novo synthesis of dTTP CDP is reduced to dCDP by ribonucleotide reductase (RNR) dCTP is synthesized by phosphorylation of dCDP Excess dCTP is hydrolyzed to dCMP by RS21-C6 dCMP is converted to dTMP by dCMP deaminase (DCTD) and thymidylate synthase (TS) dTMP is converted to dTTP by two steps of phosphorylation RS21-C6 plays a role in regulation of the dCTP ⁄ dTTP ratio in dNTP pools for nuclear and mitochondrial DNA synthesis (B) RS21-C6 may hydrolyze 5-I-dCTP or its structurally related nucleotides to prevent inappropriate CpG methylation because 500 lm ITP did not inhibit hydrolysis of dCTP by RS21-C6 in our preliminary experiment 5-I-dCTP is a derivative molecule of dCTP, in which C5 of the cytosine base is iodinated Halogenation of cytosine at C5, including chlorination and bromination, has been shown to occur under physiological conditions Such halogenation occurs in the presence of either myeloperoxidase or eosinophil peroxidase, which are produced by phagocytic cells [28,29] In these reactions, hypohalous acids, inter-halogen and haloamines are candidate intermediate molecules that can diffuse across plasma membranes into the cytoplasm and halogenate cytosine in cells Kawai et al [30] have previously detected halogenated cytosine in DNA in inflamed tissues Their results indicate in vivo halogenation of cytosine, but not provide direct evidence for intra-cellular halogenation Valinluck et al reported that 5-iodocytosine, 5-bromocytosine and 5-chlorocytosine, at a CpG site of DNA, can mimic 5-methylcytosine and induce inappropriate DNA methyltransferase 1-dependent methylation within the CpG sequence [31,32] The induction effect of 5-iodocytosine was the greatest among the 5-halogenated cytosines, and was even better than that of 5-methylcytosine 5-halogenated cytosine, at a CpG site, may enhance the binding of methyl-CpG-binding protein [33] These reports suggest that the 5-halogenated dCTP generated in chronic inflamed tissues might be incorporated into promoter regions of important genes, such as tumor-suppressor genes, and induce their silencing through inappropriate CpG methylation of the promoter regions or by binding of methyl-CpG-binding protein 2, resulting in tumorigenesis RS21-C6 may FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS 1661 A mammalian dCTPase that prefers 5-iodocytosine M Nonaka et al prevent such deleterious effects by hydrolyzing modified deoxynucleotides including 5-I-dCTP or its structurally related molecules (Fig 6B) 5-Methyl-dCTP is also a potential inducer of inappropriate CpG methylation by DNA methyltransferase when it is incorporated in a CpG site Although 5-methyl-dCMP is a poor substrate for mammalian nucleoside monophosphate kinases [34,35], 5-methyldCMP has been shown to be incorporated into the DNA of Chinese hamster ovary cells with low dCMP deaminase activity [36] Using preliminary comparative modeling, Moroz et al [8] found that 5-methyldCTP is a potential substrate candidate of RS21-C6, and 5-methyl-dCTP hydrolyzing activity of RS21-C6 was recently reported by Wu et al [10] In the present study, we show that the kcat ⁄ Km value of RS21-C6 for 5-methyl-dCTP is almost the same as that for dCTP More detailed comparisons of the enzyme kinetics for dCTP and 5-methyl-dCTP are necessary to establish the physiological role of RS21-C6 for 5-methyl-dCTP XTP3TPA (gi|13129100) is a human homolog of RS21-C6 Our study demonstrated the substrate preference of RS21-C6 for deoxynucleotides, cytosine bases and iodination at C5 of cytosine These data suggest that a defect of human XTP3TPA might cause a nuclear DNA replication block or mitochondrial DNA depletion, as a result of an imbalanced dCTP ⁄ dTTP ratio Moreover, XTP3TPA might be involved in tumorigenesis in chronically inflamed tissues, as a result of accumulation of modified deoxynucleotides Experimental procedures Synthetic oligonucleotides The synthetic oligonucleotides listed below, used as PCR primers, were purchased from Genenet Co Ltd (Fukuoka, Japan), Sigma-Aldrich Japan (Tokyo, Japan) and Takara Bio Inc (Ohtsu, Japan): 5¢Nde-mMAZG, 5¢-ATACATATG TCCACGGCTGGTGACGGTGAGCG-3¢; 5¢Nco-mMAZG, 5¢-ATACCATGGCCTCCACGGCTGGTGACGGTGAGC3¢; 3¢mMAZG-BamHI, 5¢-ATAGGATCCTTATGTGGAAG CCTGGTCTCTC-3¢; RS21-C6 forward, 5¢-GCGAGCTGGC AGAACTCTTC-3¢; RS21-C6 reverse, 5¢-TTTGGTGGCCA TGCTTGA-3¢; 18S rRNA forward, 5¢-AGGATGTGAAGG ATGGGAAG-3¢; 18S rRNA reverse, 5¢-ACGAAGGCCCC AAAAGTG-3¢ Nucleotides The nucleotides used as substrates for RS21-C6 were purchased from Sigma-Aldrich (St Louis, MO, USA), TriLink 1662 Biotechnologies Inc (San Diego, CA, USA) or Jena Bioscience (Jena, Germany) Preparation of mouse thymocyte extract Five-week-old C57BL ⁄ 6J male mice (Clea Japan, Tokyo, Japan) were dissected under pentobarbital anesthesia (75 mgỈkg)1, intraperitoneally), and killed by blood drainage from abdominal vessels The thymus was removed and ground between glass slides to prepare thymocyte suspensions Thymocytes (6 · 108) were suspended in mL of lysis buffer {25 mm 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonate-Na pH 7.2, 150 mm NaCl, 60 mm MgCl2, 0.05% Nonidet P-40 (Nacalai tesque, Kyoto, Japan), mm dithiothreitol, 1% protease inhibitor cocktail (Nacalai Tesque)}, and were disrupted by sonication Cell lysates were then centrifuged at 100 000 g for 30 The supernatant was collected as the thymocyte extract Handling and killing of all animals used in this study were in accordance with the national prescribed guidelines, and ethical approval for the studies was granted by the Animal Experiment Committee of Kyushu University (Fukuoka, Japan) Preparation of ITP-agarose ATP-agarose (adenosine 5¢-triphosphate agarose, SigmaAldrich; 25 lL bed volume) or 25 lL agarose carrier matrix were washed for twice in mL m sodium acetate buffer (pH 3.2), and then suspended in 150 lL of deamination buffer [100 mm sodium nitrite (NaNO2), 500 mm sodium thiocyanate (NaSCN), m sodium acetate (NaCH3COO) pH 3.2], and incubated at 37 °C for 60 Then each agarose aliquot was washed for twice in mL of water and used as ITP-agarose or as deaminated agarose, respectively To confirm the nucleotide immobilized on each agarose, the base moiety was excised by incubation in m HCl at 100 °C for h, and analyzed by HPLC after neutralization and filtration Purification and identification of ITP-binding proteins ITP-agarose was resuspended in 450 lL thymocyte extract in a microtube, and mixed by vertical rotation for 30 Agarose that had been subjected to deamination was used as the negative control Each agarose sample was then washed for three times in mL of lysis buffer without protease inhibitor All procedures were performed at °C Each agarose was then resuspended in 40 lL of 2· SDS sampling buffer (Sigma-Aldrich), and incubated at 95 °C for The supernatant was collected after centrifugation at 140 g for s at room temperature Proteins in each sample were separated by SDS–PAGE and analyzed by LC-MS ⁄ MS as described FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS M Nonaka et al previously [37] Collision-induced dissociation spectra were acquired and compared with those in the International Protein Index (IPI version 3.16; European Bioinformatics Institute Hinxton, UK) using the MASCOT search engine (Matrix Science, Boston, MA, USA) The high-scoring peptide sequences (MASCOT score > 45) assigned by MASCOT were manually confirmed by comparison with the corresponding collision-induced dissociation spectra Finally we selected as candidate proteins those proteins for which multiple peptides were identified in this analysis Isolation of RS21-C6 cDNA RS21-C6 cDNA fragments, RS21-C6(NdeI/BamHI) and RS21-C6(NcoI/BamHI) were amplified by PCR from a mouse fibroblast cell line, NIH/3T3, prepared as described previously [38], using primer sets 5¢Nde-mMAZG/ 3¢mMAZG-BamHI and 5¢Nco-mMAZG/3¢mMAZGBamHI Amplified fragments were subcloned into pT7Blue-2 T-vector (Novagen, Madison, WI, USA) to generate the plasmids pT7Blue2T:RS21-C6(NdeI ⁄ BamHI) and pT7Blue2T:RS21-C6(NcoI ⁄ BamHI), respectively Construction of expression plasmids Plasmids pET3a:RS21-C6, pET32a(+):RS21-C6, pcDNA3.1hyg(+):RS21-C6 and pIRES2-EGFP:RS21-C6 were prepared by inserting DNA fragments containing the ORF of RS21-C6 cDNA into the NdeI ⁄ BamHI site of pET3a (Novagen), the NcoI ⁄ BamHI site of pET32a(+) (Novagen) or the XhoI ⁄ BamHI site of pcDNA3.1hyg(+) (Invitrogen, Carlsbad, CA, USA) or into the XhoI/BamHI site of pIRES2-EGFP (Clontech Laboratories Inc., Mountain View, CA, USA), respectively Expression and purification of recombinant RS21-C6 protein Expression of recombinant RS21-C6, without any tag sequence, was induced in E coli BL21-CodonPlus(DE3)RIL cells (Stratagene, La Jolla, CA, USA) transformed with pET3a:RS21-C6, as described previously [39] Cells were suspended in buffer A (50 mm Tris ⁄ HCl pH 8.0, 100 mm NaCl, mm EDTA, mm 2-mercaptoethanol, mm phenylmethanesulfonyl fluoride, lgỈmL)1 pepstatin A, lgỈmL)1 chymostatin, lgỈmL)1 leupeptin), disrupted by sonication, and clarified by centrifugation at 20 000 g for 30 at °C Proteins in the supernatants were precipitated using ammonium sulfate (40–50% saturation), and re-dissolved in buffer B (50 mm Tris ⁄ HCl pH 8.0, 100 mm NaCl, 5% glycerol, mm MgCl2, mm 2-mercaptoehanol) Dissolved samples were dialyzed three times against L of buffer B and loaded onto HiTrap-Q HP anion exchange columns (GE Healthcare, Chalfont A mammalian dCTPase that prefers 5-iodocytosine St Giles, UK) equilibrated with buffer C (50 mm Tris ⁄ HCl pH 8.0, 50 mm NaCl, 5% glycerol, mm MgCl2, mm 2-mercaptoehanol) Binding proteins were eluted using a linear gradient of NaCl (50–1000 mm) Fractions containing RS21-C6 protein were applied onto Superdex 75 HR10 ⁄ 30 size exclusion columns (Sigma-Aldrich) equilibrated with buffer B Fractions containing RS21-C6 were then loaded onto a MonoQ HR5 ⁄ anion exchange column (GE Healthcare) equilibrated with buffer C, and eluted using a linear gradient of NaCl (50–1000 mm) Fractions containing RS21-C6 were loaded sequentially onto HiTrap-S HP and HiTrap heparin columns (GE Healthcare), and flow-through fractions were collected RS21-C6 protein was concentrated using HiTrap-Q columns, dialyzed against buffer D (50 mm Tris ⁄ HCl pH 8.0, 100 mm NaCl, 50% glycerol, mm MgCl2, mm dithiothreitol), and stored at )30 °C as purified RS21-C6 protein Nucleotide-hydrolyzing assay with RS21-C6 protein Substrate nucleotides were incubated in 18 lL of reaction buffer [50 mm Tris ⁄ HCl pH 8.0, 100 mm KCl, mm MgCl2, 100 lgỈmL)1 BSA (New England Biolabs Inc., Ipswich, MA, USA), mm dithiothreitol] at 30 °C for 10 Then, lL of 500 nm RS21-C6 protein, in reaction buffer, was added to the reaction and further incubated at 30 °C for 0–30 Sample solutions were mixed with 10 lL of ice-cold 50 mm EDTA to stop the reactions, clarified by centrifugation at 9000 g for 10 at °C, and separated on SunFire C18 lm 4.6 · 250 mm columns (Waters, Milford, MA, USA) or TSK gel DEAE-2SW columns (Tohso, Tokyo, Japan) using an Alliance photodiode array HPLC system (Waters), at a flow rate of mLỈmin)1 with HPLC buffer (0.1 m potassium phosphate pH 6.0, 5% methanol) or HPLC buffer (75 mm sodium phosphate pH 6.0, 20% acetonitrile, 0.4 mm EDTA) The amounts of nucleotide were quantified by UV absorption Anti-RS21-C6 Ig An antigen, TrxA-RS21-C6 protein, was expressed in E coli BL21-CodonPlus (DE3)-RIL cells transformed with pET32a(+):RS21-C6, and purified by metal affinity chromatography with TALON beads (Clontech) Preparation of rabbit anti-TrxA-RS21-C6 serum and affinity purification of anti-RS21-C6 Ig were performed as described previously [39,40] Western blot Western blot analysis using antibodies against anti-RS21C6, Lamin B (Santa Cruz Biotechnology Inc., Santa Cruz, FEBS Journal 276 (2009) 1654–1666 ª 2009 The Authors Journal compilation ª 2009 FEBS 1663 A mammalian dCTPase that prefers 5-iodocytosine M Nonaka et al CA, USA), HSP60 (LK-1; StressGen Biotechnologies Corp., Victoria, Canada), GAPDH (Chemicon International Inc., Temwcula, CA, USA) or using anti-ITPase serum was performed as described previously [5,39] Cell culture Mouse embryonic fibroblast cells were prepared as described previously [41] Mouse embryonic fibroblast cells, NIH ⁄ 3T3 cells and BALB ⁄ 3T3 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 unitsỈmL)1 penicillin and 100 lgỈmL)1 streptomycin A20, BW5147.3, WEHI231 and J774A.1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum, 100 unitsỈmL)1 penicillin, 100 lgỈmL)1 streptomycin and 50 lm 2-mercaptoethanol Transfection of plasmid DNA into A20 cells was performed using an MP-100 microporator (Digital Bio Technology, Seoul, Korea) Fractionation of mouse liver cells After fasting overnight, a six-month-old C57BL ⁄ 6J female mouse (Clea Japan) was dissected under pentobarbital anesthesia (75 mgỈkg)1, intraperitoneally) After draining blood by cutting abdominal vessels, the liver was removed and rinsed with 0.25 m sucrose at °C The following procedures were all performed at °C The liver was homogenized in a Teflon Potter–Elvehjem homogenizer, and fractionated by centrifugation at 700 g for 10 The pellet was rinsed with 0.25 m sucrose, and resuspended in 0.25 m sucrose as a nuclear fraction The supernatant (postnuclear supernatant) was centrifuged at 10 000 g for 10 The pellet was rinsed with 0.25 m sucrose and suspended in 0.25 m sucrose The resulting solution was used as the mitochondrial fraction The supernatant obtained after centrifugation at 10 000 g was re-centrifuged at 110 000 g for 60 The resulting supernatant was used as the cytosolic fraction Real-time quantitative PCR A 10-month-old C57BL ⁄ 6J male mouse (Clea Japan) was dissected under pentobarbital anesthesia (75 mgỈkg)1, intraperitoneally) After transcardiac perfusion with saline, tissues were removed Total RNA samples from mouse tissues except the thyroid gland were prepared using an Isogen kit (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions Thyroid gland total RNA was purchased from Clontech Total RNAs were treated using RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) at 37 °C for 60 cDNA was synthesized from lg total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, 1664 USA) using random primers in a total volume of 20 lL Real-time quantitative PCR was performed to measure the levels of RS21-C6 mRNA and 18S rRNA using an ABI Prism 7000 sequence detection system (Applied Biosystems) with 10 ng cDNA, 50 nm primers and Power SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 25 lL The PCR reaction was performed as follows: a single cycle of 50 °C for min, a single cycle of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for The primers were designed using primer express software (Applied Biosystems) and their sequences are described above The primers spanned intron and exon junctions The specificity of the PCR products was established by dissociating curve analysis, 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(Tokyo, Japan) and Takara Bio Inc (Ohtsu, Japan): 5¢Nde-mMAZG, 5¢-ATACATATG TCCACGGCTGGTGACGGTGAGCG-3¢; 5¢Nco-mMAZG, 5¢-ATACCATGGCCTCCACGGCTGGTGACGGTGAGC3¢; 3¢mMAZG-BamHI, 5¢-ATAGGATCCTTATGTGGAAG CCTGGTCTCTC-3¢;... ITP- and ATP-agarose (Fig 2C) D Agarose ATP-agarose (kDa) 25 (kDa) ITP-agarose B ITP-agaros e A ATP-agaros e Fig Preparation of ITP-agarose ATP-agarose were incubated in M HCl before (upper graph)... CCTGGTCTCTC-3¢; RS21-C6 forward, 5¢-GCGAGCTGGC AGAACTCTTC-3¢; RS21-C6 reverse, 5¢-TTTGGTGGCCA TGCTTGA-3¢; 18S rRNA forward, 5¢-AGGATGTGAAGG ATGGGAAG-3¢; 18S rRNA reverse, 5¢-ACGAAGGCCCC AAAAGTG-3¢ Nucleotides