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It is proposed here that the delayed cytotoxicity of thioguanine involves the postreplicative DNA mismatch repair system. After incorporation into DNA, the thioguanine is chemically methylated by S-adenosylmethionine to form S6-methylthioguanine. During DNA replication, the S6-methylthioguanine directs incorporation of either thymine or cytosine into the growing DNA strand, and the resultant S6-methylthioguanine-thymine pairs are recognized by the postreplicative mismatch repair system. Azathioprine, an immunosuppressant used in organ transplantation, is partly converted to thioguanine. Because the carcinogenicity of N-nitrosamines depends on formation of O6-alkylguanine in DNA, the formation of the analog S6-methylthioguanine during azathioprine treatment may partly explain the high incidence of cancer after transplantation.

In vitro, following the removal of thymine from a G⅐T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S 6 -methylthioguanine ( SMe G) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (k off 1.8 ؊ 3.6 ؋ 10 ؊5 s ؊1 ; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C⅐AP and SMe G⅐AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C⅐U and SMe G⅐T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G⅐AP sites sufficiently to allow it to remove more than one thymine from G⅐T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase ␤, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.In mammalian cells, 2-7% of the total cytosine is methylated. Spontaneous deamination of 5-methylcytosine, which is somewhat faster than cytosine (1), generates G⅐T mispairs in DNA. The repair of these G⅐T mismatches is initiated by thymine DNA glycosylase which excises the mismatched thymine (2, 3). 5-Methylcytosine occurs almost exclusively in the sequence Me CpG, and in keeping with its proposed role in the repair of G⅐T mispairs resulting from the deamination of 5-methylcytosine, thymine DNA glycosylase shows a strong preference for removal of thymine from CpG⅐T sequences (4 -7). The human enzyme has been cloned and overexpressed (8) and has been shown to belong to a family of uracil DNA glycosylases that remove uracil from G⅐U base pairs but that are distinct from the general uracil DNA glycosylase enzyme (9). Thymine DNA glycosylase removes uracil from G⅐U base pairs more rapidly than it removes thymine from G⅐T base pairs (10) and can also remove uracil from C⅐U, T⅐U, and A⅐U base pairs (7) and may therefore provide a backup function to the general uracil DNA glycosylase. The glycosylase also removes thymine from base pairs with S 6 -methylthioguanine ( SMe G) 1 that ar...

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