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.

中文翻译：

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健康和患病状态下人类 CtIP 的 DNA 结合和桥接


人类 DNA 修复因子 CtIP 有助于启动双链 DNA 断裂的切除，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 是一种天然无序的蛋白质，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，我们使用大量 DNA 结合、单分子追踪和 DNA 桥接测定来研究野生型和变异 CtIP 蛋白，以更好地定义 DNA 结合域以及与人类遗传疾病相关的突变的影响。我们的工作鉴定了 CtIP C 末端区域的单体 DNA 结合域。 CtIP 非特异性地与 DNA 结合，并且可以扩散到数千个核苷酸。在单分子磁镊实验中观察到 CtIP 介导的远距离 DNA 片段桥接。然而，我们表明单独的结合不足以实现 DNA 桥接，这还需要通过 N 端结构域进行四聚化。与 Seckel 和 Jawad 综合征相关的变异 CtIP 蛋白表现出 DNA 结合和桥接活性受损。讨论了这些发现在促进 DNA 断裂修复方面的重要性。