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J. Biol. Chem., Vol. 283, Issue 35, 23665-23670, August 29, 2008

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Mutagenic and Cytotoxic Properties of 6-Thioguanine, S⁶-Methylthioguanine, and Guanine-S⁶-sulfonic Acid^*

From the Department of Chemistry, University of California, Riverside, California 92521-0403

Thiopurine drugs, including 6-thioguanine (^SG), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of ^SG nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. ^SG in DNA can be methylated by S-adenosyl-L-methionine to give S⁶-methylthioguanine (S⁶mG) and oxidized by UVA light to render guanine-S⁶-sulfonic acid (^SO3HG). Here, we constructed single-stranded M13 shuttle vectors carrying a ^SG, S⁶mG, or ^SO3HG at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S⁶mG and ^SO3HG were highly mutagenic, which resulted in GA mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with GA transition occurring at a frequency of 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

Received for publication, May 28, 2008 , and in revised form, June 27, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01 CA101864. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ To whom correspondence should be addressed: Dept. of Chemistry-027, University of California, Riverside, CA 92521-0403. Tel.: 951-827-2700; Fax: 951-827-4713; E-mail: yinsheng.wang{at}ucr.edu.

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