Zuo S, Boorstein R J, Cunningham R P, Teebor G W
Department of Biology, New York University Graduate School of Arts and Sciences, New York 10003, USA.
Biochemistry. 1995 Sep 12;34(36):11582-90. doi: 10.1021/bi00036a034.
We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5-methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions.
我们之前通过大肠杆菌内切酶III的DNA糖基化酶活性将胞嘧啶水合物及其脱氨产物尿嘧啶水合物从DNA骨架中释放出来，证明了紫外线诱导DNA中胞嘧啶水合物的形成。随后，有报道称内切酶III介导从紫外线照射的聚（dA-dT）中释放胸腺嘧啶水合物。因此，我们询问DNA中的5-甲基胞嘧啶残基是否会像紫外线诱导胞嘧啶脱氨一样发生光水合作用并脱氨生成胸腺嘧啶水合物。用UVC照射含有5-甲基胞嘧啶的交替DNA共聚物，并与内切酶III一起孵育。没有释放出5-甲基胞嘧啶水合物。相反，发生了紫外线诱导的5-甲基胞嘧啶的非酶促释放。同样，将紫外线照射的聚（dA-dT）与内切酶III一起孵育也没有释放出胸腺嘧啶水合物；胸腺嘧啶发生了非酶促释放。5-甲基嘧啶的非酶促释放依赖于氧气，受铁离子增强并被自由基清除剂抑制。相比之下，胞嘧啶的光水合作用不依赖于氧气，并且只有少量胞嘧啶被非酶促释放。因此，DNA中交替的Pu-Py序列内的5-甲基嘧啶残基不会发生光水合作用，而是其N-糖苷键发生断裂，产生无碱基（AP）位点。无法修复此类AP位点可能解释了缺乏AP内切酶活性的大肠杆菌xthnfo突变体对紫外线的敏感性。我们认为N-糖苷键的断裂是由自由基介导的，这些自由基是通过电子从紫外线激发的三重态5-甲基嘧啶转移到基态氧和/或铁离子而形成的。
