COMPARISON OF THE EFFECTS OF UV IRRADIATION ON 5-METHYL-SUBSTITUTED AND UNSUBSTITUTED PYRIMIDINES IN ALTERNATING PYRIMIDINE-PURINE SEQUENCES IN DNA

We previously demonstrated the UV-induced formation of cytosine hydrat e in DNA and its deamination product, uracil hydrate, via their releas e from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated releas e of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Ther efore, we asked whether 5-methylcytosine residues in DNA underwent pho tohydration and deamination to thymine hydrate in analogy to UV-induce d deamination of cytosine. An alternating DNA copolymer containing 5-m ethylcytosine was irradiated with UVC and incubated with endonuclease m. No 5-methylcytosine hydrate was released. Instead, W-induced nonenz ymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymin e hydrate; nonenzymatic release of thymine occurred. Nonenzymatic rele ase of 5-methylpyrimidines was oxygen dependent, enhanced by ferric io n and inhibited by free radical scavengers. In contrast, photohydratio n of cytosine was oxygen independent, and only small amounts of cytosi ne were nonenzymatically released. Thus, 5-methylpyrimidine residues w ithin alternating Pu-Py sequences in DNA do not undergo photohydration , but instead undergo cleavage of their N-glycosyl bonds yielding abas ic (AP) sites. The inability to repair such AP sites may explain the U V sensitivity of E. coli xthnfo mutants, which lack AP endonuclease ac tivity. We suggest that N-glycosyl bond cleavage is mediated by radica l species formed via transfer of an electron from UV-excited triplet 5 -methylpyrimidines to ground state oxygen and/or ferric ions.