Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.
DNA Repair (Amst). 2013 Jul;12(7):535-40. doi: 10.1016/j.dnarep.2013.04.003. Epub 2013 May 13.
The mammalian thymine DNA glycosylase (TDG) excises 5-carboxylcytosine (5caC) when paired with a guanine in a CpG sequence, in addition to mismatched bases. Here we present a complex structure of the human TDG catalytic mutant, asparagine 140 to alanine (N140A), with a 28-base pair DNA containing a G:5caC pair at pH 4.6. TDG interacts with the carboxylate moiety of target nucleotide 5caC using the side chain of asparagine 230 (N230), instead of asparagine 157 (N157) as previously reported. Mutation of either N157 or N230 residues to aspartate has minimal effect on G:5caC activity while significantly reducing activity on G:U substrate. Combination of both the asparagine-to-aspartate mutations (N157D/N230D) resulted in complete loss of activity on G:5caC while retaining measurable activity on G:U, implying that 5caC can adopt alternative conformations (either N157-interacting or N230-interacting) in the TDG active site to interact with either of the two asparagine side chain for 5caC excision.
哺乳动物胸腺嘧啶 DNA 糖基化酶 (TDG) 在与 CpG 序列中的鸟嘌呤配对时会切除 5-羧基胞嘧啶 (5caC)，除了错配碱基。在这里，我们展示了一个人 TDG 催化突变体的复杂结构，天冬酰胺 140 到丙氨酸 (N140A)，带有一个 28 碱基对的 DNA，其中包含一个 G:5caC 对在 pH4.6。TDG 通过天冬酰胺 230 (N230) 的侧链与靶核苷酸 5caC 的羧基部分相互作用，而不是如前所述的天冬酰胺 157 (N157)。将 N157 或 N230 残基突变为天冬氨酸对 G:5caC 活性的影响最小，而对 G:U 底物的活性显著降低。将天冬酰胺到天冬氨酸的突变 (N157D/N230D) 组合导致 G:5caC 的活性完全丧失，而对 G:U 的活性保持可测量，这意味着 5caC 可以在 TDG 活性位点中采用替代构象 (N157 相互作用或 N230 相互作用) 与两种天冬酰胺侧链之一相互作用以进行 5caC 切除。
