DNA binding and bridging by human CtIP in the healthy and diseased states
DNA binding and bridging by human CtIP in the healthy and diseased states
复制标题
健康和患病状态下人类 CtIP 的 DNA 结合和桥接
DOI：
10.1101/2023.12.14.571649
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Balaji S
中科院分区：
文献类型：
--
作者：
Balaji S
人DNA修复因子CtIP有助于启动双链DNA断裂的切除，以通过同源重组进行修复，部分是通过其结合和桥接DNA分子的能力。然而，CtIP是一种天然无序蛋白，与其他DNA结合蛋白没有明显的相似性，因此这些活性的结构基础仍不清楚。在这项工作中，我们使用批量DNA结合、单分子追踪和DNA桥接测定来研究野生型和变体CtIP蛋白，以更好地定义DNA结合结构域以及与人类遗传性疾病相关的突变的影响。我们的工作确定了一个单体的DNA结合结构域的C-末端区域的CtIP。CtIP与DNA非特异性结合，可以扩散到数千个核苷酸上。在单分子磁镊实验中观察到CtIP介导的远距离DNA片段的桥接。然而，我们发现，单独的结合是不够的DNA桥接，这也需要通过N-末端结构域的四聚体。与Seckel和Jawad综合征相关的变异CtIP蛋白显示受损的DNA结合和桥接活性。这些研究结果的意义，促进DNA断裂修复的背景下进行了讨论。
The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed.