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Updated: Jun 25, 2026
Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina
Published on: August 29, 2018
Mouse RS21-C6 is a mammalian 2'-deoxycytidine 5'-triphosphate pyrophosphohydrolase that prefers 5-iodocytosine
Mari Nonaka1, Daisuke Tsuchimoto, Kunihiko Sakumi
1Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan.
Abstract:
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 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.
Insights
RS21-C6 protein hydrolyzes deoxynucleoside triphosphates, including modified forms like 5-iodo-dCTP, producing essential precursors for DNA synthesis and preventing potential DNA damage.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Nucleotides are crucial for cellular functions and can undergo modifications.
- Modified nucleotides can be harmful and require elimination from cellular pools.
- ITP, a modified nucleotide, signals potential cellular stress.
Purpose of the Study:
- To identify proteins that bind to inosine triphosphate (ITP).
- To characterize the enzymatic activity of the identified RS21-C6 protein.
- To investigate the role of RS21-C6 in nucleotide metabolism and DNA integrity.
Main Methods:
- Protein screening for ITP-binding activity.
- Purification and characterization of recombinant RS21-C6 protein.
- Enzymatic assays measuring pyrophosphohydrolase activity on various nucleotides.
Main Results:
- RS21-C6 protein was identified and found to bind ITP and ATP.
- Recombinant RS21-C6 exhibits pyrophosphohydrolase activity, preferring deoxynucleoside triphosphates and cytosine bases.
- RS21-C6 efficiently hydrolyzes dCTP and 5-I-dCTP, with a higher affinity for the modified form.
- The enzyme produces dCMP from dCTP, a precursor for dTTP synthesis.
Conclusions:
- RS21-C6 generates dCMP from dCTP, supporting de novo dTTP synthesis.
- RS21-C6 may protect cells from DNA damage by hydrolyzing modified dCTP, preventing mutagenesis and replication errors.
