Zhao Xiaobei, Muller James G, Halasyam Mohan, David Sheila S, Burrows Cynthia J
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, USA.
Biochemistry. 2007 Mar 27;46(12):3734-44. doi: 10.1021/bi062214k. Epub 2007 Feb 27.
Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3' end of the nick compared to the 5' end and to lesions located in the intact template strand. In general, substrates containing the OG.A mismatch were more readily ligated than those with the OG.C mismatch. Ligations of duplexes containing the OA.T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI.A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG.A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG.C-, OI.C-, OI.A-, and OA.T-containing duplexes compared to that of a G.C-containing duplex. Substrates containing Gh.A, Gh.G, Sp.A, and Sp.G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G.C base pair, while the Km value for OG.A was 5 times lower than that for G.C.
连接酶通过在切除受损核苷酸并重新插入正确核苷酸后封闭单链切口来完成DNA损伤修复的最后阶段。根据具体情况和修复过程的成功与否，损伤可能会留在连接位点，要么在模板中，要么在待连接的寡聚物末端。使用噬菌体T4 DNA连接酶进行连接实验，在总共96种不同的双链体中，在切口位点周围的四个位置存在嘌呤损伤。如预期的那样，氧化损伤8-氧代-7,8-二氢鸟苷（OG）表明，与5'端以及完整模板链中的损伤相比，该酶对切口3'端的损伤最为敏感。一般来说，含有OG.A错配的底物比含有OG.C错配的底物更容易连接。含有可以采用反-反构象的OA.T碱基对（OA = 8-氧代-7,8-二氢腺苷）的双链体的连接效率很高。含有OI.A错配的双链体（OI = 8-氧代-7,8-二氢次黄嘌呤）的行为与OG.A相似。由于其还原电位低，OG很容易被氧化为二级氧化产物，如胍基乙内酰脲（Gh）和螺环亚氨基二氢脲（Sp）核苷；这些损伤在嘌呤原来的C8位置也含有一个氧代基团。当与A和G相对时，含有Gh和Sp的寡聚物会发生连接，尽管总体连接效率比大多数OG碱基对低得多。对模板中损伤的代表性例子进行了稳态动力学研究。与含有G.C的双链体相比，含有OG.C-、OI.C-、OI.A-和OA.T的双链体的Km增加了90-100倍。含有Gh.A、Gh.G、Sp.A和Sp.G碱基对的底物的Km值比含有G.C碱基对的底物高20-70倍，而OG.A的Km值比G.C低5倍。
