Molecular Biophysics and Structural Biology Graduate Program, University of Pittsburg, Pittsburgh, PA, USA.
UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA.
Nat Commun. 2024 Oct 25;15(1):9226. doi: 10.1038/s41467-024-53497-7.
Base excision repair is the main pathway involved in active DNA demethylation. 5-formylcytosine 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 study TDG in the presence and absence of 5-formylcytosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of base modifications. TDG active site variants and truncated N-terminus, reveals these variants alter base modification search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG is found to either bypass, colocalize with, or encounter but not bypass the nucleosome. Truncating the N-terminus reduces the number of interactions with the nucleosome. Our findings provide mechanistic insights into how TDG searches for modified DNA bases 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种氧化部分，被胸腺嘧啶 DNA 糖基化酶 (TDG)识别并去除，从而产生无碱基位点。我们使用单分子荧光实验研究了有和没有 5-甲酰基胞嘧啶存在时的 TDG。TDG 表现出多种线性扩散模式，包括跳跃和滑动，以寻找碱基修饰。TDG 活性位点变体和截断的 N 端揭示了这些变体改变了 TDG 的碱基修饰搜索和识别机制。在含有未损伤核小体的 DNA 上，TDG 要么绕过、与核小体共定位，要么遇到但不绕过核小体。截断 N 端会减少与核小体的相互作用次数。我们的发现为 TDG 如何在染色质中搜索修饰的 DNA 碱基提供了机制上的见解。
