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J. Biol. Chem., Vol. 279, Issue 17, 17019-17026, April 23, 2004

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Mitochondrial Deoxyribonucleotides, Pool Sizes, Synthesis, and Regulation^*

Chiara Rampazzo, Paola Ferraro, Giovanna Pontarin, Sonia Fabris, Peter Reichard, and Vera Bianchi

From the Department of Biology, University of Padova, I-35131 Padova, Italy

We quantify cytosolic and mitochondrial deoxyribonucleoside triphosphates (dNTPs) from four established cell lines using a recently described method for the separation of cytosolic and mitochondrial (mt) dNTPs from as little as 10 million cells in culture (Pontarin, G., Gallinaro, L., Ferraro, P., Reichard, P., and Bianchi, V. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12159–12164). In cycling cells the concentrations of the phosphates of thymidine, deoxycytidine, and deoxyadenosine (combining mono-, di-, and triphosphates in each case) did not differ significantly between mitochondria and cytosol, whereas deoxyguanosine phosphates were concentrated to mitochondria. We study the source and regulation of the mt dTTP pool as an example of mt dNTPs. We suggest two pathways as sources for mt dTTP: (i) import from the cytosol of thymidine diphosphate by a deoxynucleotide transporter, predominantly in cells involved in DNA replication with an active synthesis of deoxynucleotides and (ii) import of thymidine followed by phosphorylation by the mt thymidine kinase, predominantly in resting cells. Here we demonstrate that the second pathway is regulated by a mt 5'-deoxyribonucleotidase (mdN). We modify the in situ activity of mdN and measure the transfer of radioactivity from [³H]thymidine to mt thymidine phosphates. In cycling cells lacking the cytosolic thymidine kinase, a 30-fold overproduction of mdN decreases the specific radioactivity of mt dTTP to 25%, and an 80% decrease of mdN by RNA interference increases the specific radioactivity 2-fold. These results suggest that mdN modulates the synthesis of mt dTTP by counteracting in a substrate cycle the phosphorylation of thymidine by the mt thymidine kinase.

Received for publication, December 22, 2003 , and in revised form, January 22, 2004.

^* This work was supported by European Commission Grant QLRT-CT-2000-01004, Italian Association for Cancer Research (AIRC) and Telethon Italia Grant GP0140Y01 (to V. B.). 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.

To whom correspondence should be addressed. Tel.: 39-0498276282; Fax: 39-0498276280; E-mail: vbianchi{at}mail.bio.unipd.it.

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