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
Trang 15¢-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
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
exam-ple, 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 mole-cules, 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
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 2 September 2008, revised
8 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 biolo-gical 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 hydro-lyzed several canonical nucleoside triphosphates to the corresponding nucleoside monophosphates The pyrophosphohydrolase activity of RS21-C6 showed a preference for deoxynucleoside triphosphates and cytosine bases The kcat⁄ Km (s)1Æm)1) values were 3.11· 104, 4.49· 103 and 1.87· 103for 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⁄ Kmvalue compared with that of its corre-sponding unmodified nucleotide, dCTP RS21-C6 is expressed in both pro-liferating and non-propro-liferating 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 More-over, RS21-C6 may also prevent inappropriate DNA methylation, DNA replication blocking or mutagenesis by hydrolyzing modified dCTP
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¢-5-bromo-2¢-deoxycytidine 5¢-triphosphate; 5-I-dCTP, 5-iodo-2¢-5-bromo-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.
Trang 2Some 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
degenera-tive diseases in humans [1] Normal functions of
nucle-otides, 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)gua-nosine 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,
MTH1-null 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
gener-ates 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
hypo-xanthine and hypo-xanthine can mis-pair with cytosine or
thymine, respectively Recently, mammalian inosine
triphosphate pyrophosphohydrolases (ITPases) have
been reported to hydrolyze deaminated purine
nucleo-side triphosphates to the corresponding nucleonucleo-side
monophosphates and pyrophosphates [5,6]
ITPase-null 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
synthe-sis, 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-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 bac-terial NTP pyrophosphatase MazG, it has been described as a member of the all-a NTP pyrophospho-hydrolase 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 recom-binant 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⁄ Kmvalue 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 hypo-xanthine 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 Quan-tification 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 thymo-cyte 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 4 and 5, respectively Specific binding of ITPase to ITP-agarose was confirmed by western blot analysis of pulldown samples using anti-ITPase 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 pre-pared as described in Experimental procedures We performed pulldown experiments, using ITP-agarose
Trang 3from cell extracts of Escherichia coli BL21-CodonPlus (DE3)-RIL that had been transformed with pET3a: RS21-C6 and induced for RS21-C6 expression Recom-binant RS21-C6 protein in bacterial cell extracts also bound to both ITP- and ATP-agarose (Fig 2C)
0.00
0.02
0.04
0.06
0
Before deamination
0.00
0.02
0.04
0.06
0
Retention time (min)
*
After deamination
Fig 1 Preparation of ITP-agarose ATP-agarose were incubated in
1 M HCl before (upper graph) or after (lower graph) deamination,
and the bases released were analyzed by HPLC The peaks
indi-cated by an open arrowhead and a closed arrowhead were
com-pared with peaks of standard samples, and were identified as
adenine base and hypoxanthine base, respectively The peak
indi-cated 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).
25 7
17 4
(kDa )
ITPase 25.7
ITP-agarose ATP-agarose Agarose
100
75
37
25
20
15
50 (kDa)
Recombinant RS21-C6
20
15
25 (kDa) ATP-agarose ITP-agarose Agarose
Recombinant RS21-C6
1
2
3
4
6
7
8
9
10
(kDa)
25.7
17.4
31.6 47.3
84.7
114
D
B
C
A
Fig 2 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 · 10 7 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 · 10 7
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.
Table 1 ITP-binding proteins identified by LC-MS ⁄ MS analysis Band
no Protein description
NCBInr accession no.
1 Acetyl coenzyme A acetyltransferase
1 precursor
gi|21450129
2 Glyceraldehyde-3-phosphate
dehydrogenase
gi|50233866
Similar to ISOC2 protein gi|20818892
Phospholipid hydroperoxide glutatione peroxidase
gi|2522259
Divalent cation tolerant protein CUTA isoform 1
gi|62198210
7 Unidentified
8 Isochorismatase domain-containing 1 gi|31541909
10 Unidentified
Trang 4TrxA-RS21-C6 protein was expressed from
pET32a(+):RS21-C6 and was used as an antigen to
prepare anti-RS21-C6 rabbit serum Western blot
anal-ysis, using affinity-purified anti-RS21-C6 Ig, showed
that endogenous RS21-C6 also binds to both ITP- and
ATP-agarose (Fig 2B, lower panel)
Glyceraldehyde-3-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
Expression of endogenous RS21-C6 Western blot analysis using anti-RS21-C6 Ig and whole-cell extracts from several mouse cell lines pro-duced 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 plas-mids 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 approxi-mately 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 sam-ples, indicating that anti-RS21-C6 specifically reacts
25.7
17.4
(kDa) 20 pcDNA pcDNA :RS21-C6 pIRES2-EGFP pIRES2-EGFP :RS21-C6
15
(kDa)
RS21-C6
GAPDH
RS21-C6
GAPDH
31.3 47.3
84.7
114
W N Mt Cy t
RS21-C6 Lamin B HSP60
GAPDH
0
5
10
15
20
Cerebrum Cerebellum
Eye Spinal cord
Heart Kidney Liver Lung
Esophagus Lymph node Spleen Bone marrow
Thymus Testis Uterus
Stomach Salivary gland Ovary
Skeletal muscle Thyroid gland
C
D
Fig 3 Expression of endogenous RS21-C6.
(A) Western blot analysis of RS21-C6
pro-tein in various mouse cell lines A whole-cell
extract from 1.0 · 10 5 cells of each cell line
was loaded in each lane Proteins were
sep-arated by SDS–PAGE, and then transferred
to a poly(vinylidene difluoride) membrane.
Signals for RS21-C6 protein and of GAPDH
were detected using RS21-C6 and
anti-GAPDH 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
vec-tors by electroporation The cells were
incu-bated for 24 h, and whole-cell extracts from
1.0 · 10 5 cells were loaded on each lane.
(C) Intracellular localization of RS21-C6
pro-tein 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,
respec-tively (D) Real-time quantitative PCR
analy-sis 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.
Trang 5with 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 ubiqui-tously expressed and that expression was highest in the liver and heart, and to a lesser extent the salivary gland (Fig 3D)
Nucleoside triphosphate pyrophosphohydrolase activity of RS21-C6 protein
In a preliminary analysis, using canonical nucleotides, purified RS21-C6 protein showed strong pyrophospho-hydrolase activity on dCTP, producing dCMP (Fig 4A, middle panel) We also analyzed the reaction product using BIOMOL GREEN reagent (Enzo Bio-chem, 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 pyrophos-phohydrolase activity of RS21-C6 using dCTP as a substrate RS21-C6 showed a temperature-dependent increase of activity up to 60C (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 MgCl2and MnCl2 No activity was detected in reactions without added metals, and maximum activity was measured in reactions containing 100 mm MgCl2 At 100 mm, MnCl2did not support full activity (Fig 4D) RS21-C6 showed the same activity with various concentrations
of KCl between 0 and 1000 mm (Fig 4E) NaCl mod-erately reduced RS21-C6 activity Based on these results and in view of physiological conditions, we
0.08
0.04
0.00
0.08
0.04
0.00
0.08
0.04
0.00
Retention time (min)
dCTP + buffer
dCTP + RS21-C6
dCMP standard
0
20
40
60
80
100
Temperature (°C)
0
20
40
60
80
100
pH
PIPES-Na
Tris-HCl
AMPD-HCl
0
20
40
60
80
100
100
Mg 2+
Mn 2+
0
20
40
60
80
100
Salt concentration (m M )
Metal ion concentration (m M )
NaCl KCl
A
B
C
D
E
Fig 4 dCTP pyrophosphohydrolase activity of RS21-C6 protein (A) Hydrolysis of dCTP to dCMP by RS21-C6 Substrate dCTP (300 l M ) was incubated for 20 min in reaction buffer supplemented with
50 n M of purified RS21-C6 protein Reaction products were ana-lyzed 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 ana-lyzed 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.
Trang 6performed 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
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 2 Except for CTP, RS21-C6 did not hydrolyze the analyzed ribo-nucleotides, even ITP For the analyzed deoxynu-cleotides, 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 nucleo-tides, 5-I-dCTP had an eightfold higher kcat⁄ Km value compared to its corresponding unmodified deoxy-nucleotide, 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 5 The oligomeric structure of RS21-C6, together with the conformational flexibility of its active sites, suggests cooperativity between the sites and allo-steric regulation However, we obtained a typical Michaelis–Menten-type saturation curve rather than the sigmoid curve typical of non-Michaelis–Menten-type 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
Table 2 Kinetic parameters for nucleoside triphosphate
pyrophos-phohydrolase activity of RS21-C6 protein.
Substrate K m (l M ) k cat (s)1) k cat ⁄ K m (s)1Æ M )1)
2-OH-ATP No activity
8-oxo-dGTP No activity
0 10 20 30 40
50
60 70 [Substrate] (µ M )
0 1 2 3
–1 )
2 µ M
3.8 µ M
7.8 µ M
15.6 µ M
31.3 µ M
62.5 µ M
125 µ M
250 µ M
500 µ M
1000 µ M
0 5 10
30
0
60
A
B
[Substrate] –1 (µ M–1)
0.4 0.2
50
25
C
Fig 5 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 l M
(A) Enzyme reaction curves (B) Michaelis–
Menten plot using the results at substrate
concentrations from 0 to 62.5 l M , showing
the saturation pattern (C) A Lineweaver–
Burk plot prepared using all the results.
Trang 7We 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
triphos-phate 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
tet-ramer [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
vari-ous dUTPases [Campylobacter jejuni dUTPase
(CjdUTPase), Leishmania major dUTPase,
Trypanoso-ma cruzi dUTPase (TcdUTPase)], dCTPases
(entero-bacteria phage T2 dCTPase, entero(entero-bacteria 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
TcdUT-Pase, the residues corresponding to Asn125 of
RS21-C6, bind to the 2¢-deoxyribose moiety of substrates
This residue is not conserved in either the HisE family,
which has phosphoribosyl-ATP pyrophosphatase
activ-ity (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
fami-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 anal-ysis of the crystal structure of RS21-C6 with the sub-strate They used the core fragment of RS21-C6 (RSCUT: residues 21–126) in which the Asn125 resi-due is located very close to the C-terminal end There-fore, 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 gluta-mine 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, halogena-tion at the C5 posihalogena-tion, particularly iodinahalogena-tion, signifi-cantly increased its affinity to RS21-C6 It is possible that one of the residues that form the substrate-bind-ing pocket may recognize the iodine at the C5 posi-tion 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 mamma-lian 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 con-verted to dTTP by a two-step phosphorylation reac-tion 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 experi-ments using [5-3H]cytidine, Bianchi et al [20] estimated that dCTP was incorporated into DNA at a rate of
16 pmolÆmin)1 in rapidly growing mouse 3T6 cells, and that dTMP was formed from free dCMP at a rate
Trang 8of 10 pmolÆmin)1 A defect in dCMP deaminase in
hamster fibroblasts was reported to cause an
imbal-anced dCTP⁄ dTTP ratio, and to mildly affect the
fidelity of DNA replication [21] dCTP is not an
effector molecule that allosterically controls
ribonucle-otide reductase (RNR) Therefore, cells must have
other mechanisms to regulate the cytosolic dCTP
concentration In this regard, dCTP
pyrophosphohy-drolase plays an important role in avoiding
accumu-lation 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
local-ized 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
cyto-plasm 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
deple-tion caused by mutadeple-tions 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]
Simi-larly 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,
phos-phorylated 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-can-onical nucleotides from the intracellular NTP pool
Here, we have shown that RS21-C6 has the highest
kcat⁄ Km value for the modified deoxynucleotide
5-I-dCTP 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,
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 bromina-tion, has been shown to occur under physiological con-ditions Such halogenation occurs in the presence of either myeloperoxidase or eosinophil peroxidase, which are produced by phagocytic cells [28,29] In these reac-tions, 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 pre-viously detected halogenated cytosine in DNA in inflamed tissues Their results indicate in vivo haloge-nation of cytosine, but do 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 meth-yltransferase 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 2 [33] These reports suggest that the 5-halogenated dCTP generated in chronic inflamed tissues might be incorpo-rated 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-bind-ing protein 2, resultmethyl-CpG-bind-ing in tumorigenesis RS21-C6 may
Fig 6 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.
Trang 9prevent such deleterious effects by hydrolyzing
modi-fied deoxynucleotides including 5-I-dCTP or its
struc-turally related molecules (Fig 6B)
5-Methyl-dCTP is also a potential inducer of
inap-propriate CpG methylation by DNA
methyltransfer-ase 1 when it is incorporated in a CpG site Although
5-methyl-dCMP is a poor substrate for mammalian
nucleoside monophosphate kinases [34,35],
5-methyl-dCMP has been shown to be incorporated into the
DNA of Chinese hamster ovary cells with low dCMP
deaminase activity [36] Using preliminary
compara-tive modeling, Moroz et al [8] found that
5-methyl-dCTP 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
pref-erence 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¢-ATACCATGGCCTCCACGGCTGGTGACGGTGAGC-3¢; 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
pur-chased from Sigma-Aldrich (St Louis, MO, USA), TriLink
Biotechnologies Inc (San Diego, CA, USA) or Jena Biosci-ence (Jena, Germany)
Preparation of mouse thymocyte extract Five-week-old C57BL⁄ 6J male mice (Clea Japan, Tokyo, Japan) were dissected under pentobarbital anesthesia (75 mgÆkg)1, intraperitoneally), and killed by blood drai-nage from abdominal vessels The thymus was removed and ground between glass slides to prepare thymocyte sus-pensions Thymocytes (6· 108
) were suspended in 3 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), 1 mm dithiothreitol, 1% protease inhibitor cocktail (Nacalai Tesque)}, and were disrupted by sonication Cell lysates were then centrifuged at 100 000 g for 30 min The super-natant was collected as the thymocyte extract Handling and killing of all animals used in this study were in accor-dance with the national prescribed guidelines, and ethical approval for the studies was granted by the Animal Experi-ment Committee of Kyushu University (Fukuoka, Japan)
Preparation of ITP-agarose ATP-agarose (adenosine 5¢-triphosphate agarose, Sigma-Aldrich; 25 lL bed volume) or 25 lL agarose carrier matrix were washed for 2 min twice in 1 mL 3 m sodium acetate buffer (pH 3.2), and then suspended in 150 lL of deamina-tion buffer [100 mm sodium nitrite (NaNO2), 500 mm sodium thiocyanate (NaSCN), 3 m sodium acetate (NaCH
3-COO) pH 3.2], and incubated at 37C for 60 min Then each agarose aliquot was washed for 2 min twice in 1 mL
of water and used as ITP-agarose or as deaminated aga-rose, respectively To confirm the nucleotide immobilized
on each agarose, the base moiety was excised by incubation
in 1 m HCl at 100C for 1 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 min Agarose that had been subjected to deamination was used as the negative control Each agarose sample was then washed for 1 min three times in 1 mL of lysis buffer without protease inhibitor All procedures were per-formed at 4C Each agarose was then resuspended in
40 lL of 2· SDS sampling buffer (Sigma-Aldrich), and incubated at 95C for 5 min The supernatant was col-lected after centrifugation at 140 g for 5 s at room temperature Proteins in each sample were separated by SDS–PAGE and analyzed by LC-MS⁄ MS as described
Trang 10previously [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¢mMAZG-BamHI 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,
pcDNA3.1-hyg(+):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.,
Moun-tain 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, 5 mm EDTA, 5 mm 2-mercaptoethanol,
1 mm phenylmethanesulfonyl fluoride, 1 lgÆmL)1
pepsta-tin A, 1 lgÆmL)1 chymostatin, 1 lgÆmL)1 leupeptin),
dis-rupted by sonication, and clarified by centrifugation at
20 000 g for 30 min at 4C Proteins in the supernatants
were precipitated using ammonium sulfate (40–50%
satura-tion), and re-dissolved in buffer B (50 mm Tris⁄ HCl
pH 8.0, 100 mm NaCl, 5% glycerol, 5 mm MgCl2, 5 mm
2-mercaptoehanol) Dissolved samples were dialyzed three
times against 1 L of buffer B and loaded onto HiTrap-Q
HP anion exchange columns (GE Healthcare, Chalfont
St Giles, UK) equilibrated with buffer C (50 mm Tris⁄ HCl
pH 8.0, 50 mm NaCl, 5% glycerol, 5 mm MgCl2, 5 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⁄ 5 anion exchange column (GE Health-care) 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, 5 mm MgCl2, 1 mm dithiothreitol), and stored at)30 C as puri-fied 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, 5 mm MgCl2, 100 lgÆmL)1 BSA (New England Biolabs Inc., Ipswich, MA, USA), 1 mm dithiothreitol] at 30C for
10 min Then, 2 lL of 500 nm RS21-C6 protein, in reac-tion buffer, was added to the reacreac-tion and further incu-bated at 30C for 0–30 min 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 min
at 4C, and separated on SunFire C18 5 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 1 mLÆmin)1 with HPLC buffer 1 (0.1 m potassium phosphate pH 6.0, 5% methanol) or HPLC buffer 2 (75 mm sodium phosphate pH 6.0, 20% acetoni-trile, 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 chro-matography 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-RS21-C6, Lamin B (Santa Cruz Biotechnology Inc., Santa Cruz,