Mouse RS21-C6 is a mammalian 2′-deoxycytidine 5′-triphosphate pyrophosphohydrolase that prefers 5-iodocytosine

Free nucleotides in living cells play important roles in a variety of biological reactions, and often undergo chemical modi. cations 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 RS21-C6 showed a preference for deoxynucleoside triphosphates and cytosine bases. The k(cat)/K-m (s(-1).M-1) values were 3.11 X 10(4), 4.49 x 10(3) and 1.87 x 10(3) 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 k(cat)/K-m 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.