Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.
J Mol Biol. 2013 Mar 25;425(6):971-6. doi: 10.1016/j.jmb.2013.01.013. Epub 2013 Jan 19.
The mammalian thymine DNA glycosylase (TDG) excises the mismatched base, uracil, thymine or 5-hydroxymethyluracil (5hmU), as well as removes 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) when paired with a guanine. In the previously solved structure of TDG in complex with DNA containing 5caC, the side chain of asparagine 157 (N157) contacts the 5-carboxyl moiety of 5caC via a weak hydrogen bond. We examined the role of N157 in recognition of 5caC by mutagenesis. The asparagine-to-alanine (N157A) mutant has no detectable base excision activity for a G:T mismatch, and its excision activity is reduced for other substrates including G:5caC. Unexpectedly, the asparagine-to-aspartate (N157D) mutant has a comparable base excision rate for G:5caC substrate to that of wild type, but it only has residual activity for G:U and no detectable activity for other substrates. We further show that the N157D mutant has higher activity for 5caC at a lower pH (6.0), suggesting that increased protonation of the carboxylate of 5caC and the aspartate facilitates base excision. The N157D mutant remains highly specific for 5caC even in the presence of large excess of genomic DNA, a property that can potentially be used for mapping the very low amount of 5caC in genomes.
哺乳动物胸腺嘧啶 DNA 糖基化酶 (TDG) 能切除错配碱基，如尿嘧啶、胸腺嘧啶或 5-羟甲基尿嘧啶（5hmU），并在与鸟嘌呤配对时，去除 5-甲酰胞嘧啶（5fC）和 5-羧基胞嘧啶（5caC）。在之前解决的 TDG 与含有 5caC 的 DNA 复合物结构中，天冬酰胺 157（N157）的侧链通过弱氢键与 5caC 的 5-羧基部分相互作用。我们通过突变研究了 N157 在识别 5caC 中的作用。天冬酰胺到丙氨酸（N157A）突变体对 G:T 错配没有可检测的碱基切除活性，其对其他底物的切除活性降低，包括 G:5caC。出乎意料的是，天冬酰胺到天冬氨酸（N157D）突变体对 G:5caC 底物的碱基切除速率与野生型相当，但对 G:U 只有残留活性，对其他底物没有可检测的活性。我们进一步表明，N157D 突变体在较低 pH（6.0）下对 5caC 的活性更高，这表明 5caC 羧酸盐和天冬氨酸的质子化增加有助于碱基切除。即使在存在大量基因组 DNA 的情况下，N157D 突变体对 5caC 仍保持高度特异性，这一特性可用于绘制基因组中非常低含量的 5caC。
