从氧化损伤的DNA中立体选择性切除胸腺嘧啶二醇
Stereoselective excision of thymine glycol from oxidatively damaged DNA.
作者信息
Miller Holly, Fernandes Andrea S, Zaika Elena, McTigue Monica M, Torres M Cecilia, Wente Maryann, Iden Charles R, Grollman Arthur P
机构信息
Laboratory of Chemical Biology, State University of New York, Stony Brook, NY, USA.
出版信息
Nucleic Acids Res. 2004 Jan 15;32(1):338-45. doi: 10.1093/nar/gkh190. Print 2004.
DNA damage created by reactive oxygen species includes the prototypic oxidized pyrimidine, thymine glycol (Tg), which exists in oxidatively damaged DNA as two diastereoisomeric pairs. In Escherichia coli, Saccharomyces cerevesiae and mice, Tg is preferentially excised by endonuclease III (Endo III) and endonuclease VIII (Endo VIII), yNTG1 and yNTG2, and mNTH and mNEIL1, respectively. We have explored the ability of these DNA glycosylases to discriminate between Tg stereoisomers. Oligonucleotides containing a single, chromatographically pure (5S,6R) or (5R,6S) stereoisomer of Tg were prepared by oxidation with osmium tetroxide. Steady-state kinetic analyses of the excision process revealed that Endo III, Endo VIII, yNTG1, mNTH and mNEIL1, but not yNTG2, excise Tg isomers from DNA in a stereoselective manner, as reflected in the parameter of catalytic efficiency (kcat/Km). When DNA glycosylases occur as complementary pairs, failure of one or both enzymes to excise their cognate Tg stereoisomer from oxidatively damaged DNA could have deleterious consequences for the cell.
活性氧产生的DNA损伤包括典型的氧化嘧啶——胸苷乙二醇（Tg），它在氧化损伤的DNA中以两个非对映异构体对的形式存在。在大肠杆菌、酿酒酵母和小鼠中，Tg分别优先被核酸内切酶III（Endo III）和核酸内切酶VIII（Endo VIII）、yNTG1和yNTG2、mNTH和mNEIL1切除。我们研究了这些DNA糖基化酶区分Tg立体异构体的能力。通过四氧化锇氧化制备了含有单一色谱纯（5S,6R）或（5R,6S）Tg立体异构体的寡核苷酸。切除过程的稳态动力学分析表明，Endo III、Endo VIII、yNTG1、mNTH和mNEIL1（但不包括yNTG2）以立体选择性方式从DNA中切除Tg异构体，这在催化效率（kcat/Km）参数中得到体现。当DNA糖基化酶以互补对形式存在时，一种或两种酶无法从氧化损伤的DNA中切除其同源Tg立体异构体可能会对细胞产生有害影响。
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