Schnable Brittani L, Schaich Matthew A, Roginskaya Vera, Leary Liam P, Weaver Tyler M, Freudenthal Bret D, Drohat Alexander C, Houten Bennett Van
bioRxiv. 2024 Jul 27:2023.10.04.560925. doi: 10.1101/2023.10.04.560925.
Base excision repair is the main pathway involved in active DNA demethylation. 5-formylctyosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we studied TDG in the presence and absence of 5-formylctyosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. We probed TDG active site variants and truncated N-terminus revealing how these variants alter the lesion search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG was found to either bypass, colocalize with, or encounter but not bypass the nucleosome. However, truncating the N-terminus reduced the number of interactions with the nucleosome. Our findings provide unprecedented mechanistic insights into how TDG searches for DNA lesions in chromatin.
碱基切除修复是参与主动DNA去甲基化的主要途径。5-甲酰基胞嘧啶和5-羧基胞嘧啶是甲基化胞嘧啶的两个氧化部分，它们被胸腺嘧啶DNA糖基化酶（TDG）识别并去除，从而产生一个无碱基位点。利用单分子荧光实验，我们研究了存在和不存在5-甲酰基胞嘧啶时的TDG。TDG在寻找损伤位点时表现出多种线性扩散模式，包括跳跃和滑动。我们探测了TDG活性位点变体和截短的N端，揭示了这些变体如何改变TDG的损伤搜索和识别机制。在含有未受损核小体的DNA上，发现TDG要么绕过核小体、与核小体共定位，要么遇到但不绕过核小体。然而，截短N端会减少与核小体的相互作用数量。我们的研究结果为TDG如何在染色质中搜索DNA损伤提供了前所未有的机制见解。
