Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, United States.
Biomolecular NMR Center, Johns Hopkins University, Baltimore, MD 21218, United States.
DNA Repair (Amst). 2020 Feb;86:102764. doi: 10.1016/j.dnarep.2019.102764. Epub 2019 Dec 10.
Many human DNA repair proteins have disordered domains at their N- or C-termini with poorly defined biological functions. We recently reported that the partially structured N-terminal domain (NTD) of human uracil DNA glycosylase 2 (hUNG2), functions to enhance DNA translocation in crowded environments and also targets the enzyme to single-stranded/double-stranded DNA junctions. To understand the structural basis for these effects we now report high-resolution heteronuclear NMR studies of the isolated NTD in the presence and absence of an inert macromolecular crowding agent (PEG8K). Compared to dilute buffer, we find that crowding reduces the degrees of freedom for the structural ensemble, increases the order of a PCNA binding motif and dramatically promotes binding of the NTD for DNA through a conformational selection mechanism. These findings shed new light on the function of this disordered domain in the context of the crowded nuclear environment.
许多人类 DNA 修复蛋白在其 N 端或 C 端具有无规则结构域，其生物学功能定义不明确。我们最近报道称，人尿嘧啶 DNA 糖基化酶 2（hUNG2）的部分结构域（NTD）可增强在拥挤环境中的 DNA 易位，还可将酶靶向单链/双链 DNA 连接点。为了了解这些影响的结构基础，我们现在报告了在存在和不存在惰性大分子拥挤剂（PEG8K）的情况下，对分离的 NTD 的高分辨率异核 NMR 研究。与稀缓冲液相比，我们发现拥挤会降低结构整体的自由度，增加 PCNA 结合基序的有序性，并通过构象选择机制极大地促进 NTD 与 DNA 的结合。这些发现为在拥挤的核环境中，该无序结构域的功能提供了新的见解。
