Proper repair of damaged DNA is critical for the maintenance of genome stability. A complex composed of Integrator subunit 3 (Ints3), single-stranded DNA-binding protein 1 (SSB1) and SSB-interacting protein 1 (SSBIP1) is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ataxia-telangiectasia mutated (ATM)-dependent signaling pathways. It is known that in this complex the Ints3 N-terminal domain scaffolds SSB1 and SSBIP1. However, the molecular basis for the function of the Ints3 C-terminal domain remains unclear. Here, we present the crystal structure of the Ints3 C-terminal domain, uncovering a HEAT-repeat superhelical fold. Using structure and mutation analysis, we show that the C-terminal domain exists as a stable dimer. A basic groove and a cluster of conserved residues on two opposite sides of the dimer bind single-stranded RNA/DNA (ssRNA/ssDNA) and Integrator complex subunit 6 (Ints6), respectively. Dimerization is required for nucleic acid binding, but not for Ints6 binding. Additionally, in vitro experiments using HEK 293T cells demonstrate that Ints6 interaction is critical for maintaining SSB1 protein level. Taken together, our findings establish the structural basis of a multifunctional Ints3 C-terminal module, allowing us to propose a novel mode of nucleic acid recognition by helical repeat protein and paving the way for future mechanistic studies.

中文翻译：

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人类Ints3 C端结构域多功能作用的结构基础


受损 DNA 的正确修复对于维持基因组稳定性至关重要。双链断裂 (DSB) 和共济失调的有效同源重组依赖性修复需要由整合子亚基 3 (Ints3)、单链 DNA 结合蛋白 1 (SSB1) 和 SSB 相互作用蛋白 1 (SSBIP1) 组成的复合物-毛细血管扩张突变（ATM）依赖性信号通路。众所周知，在该复合物中，Ints3 N 端结构域支架 SSB1 和 SSBIP1。然而，Ints3 C 末端结构域功能的分子基础仍不清楚。在这里，我们展示了 Ints3 C 端结构域的晶体结构，揭示了 HEAT 重复超螺旋折叠。通过结构和突变分析，我们表明 C 端结构域以稳定的二聚体形式存在。二聚体两侧的基本凹槽和保守残基簇分别结合单链 RNA/DNA (ssRNA/ssDNA) 和整合复合物亚基 6 (Ints6)。核酸结合需要二聚化，但 Ints6 结合不需要二聚化。此外，使用 HEK 293T 细胞的体外实验表明，Ints6 相互作用对于维持 SSB1 蛋白水平至关重要。总而言之，我们的研究结果建立了多功能 Ints3 C 端模块的结构基础，使我们能够提出一种通过螺旋重复蛋白识别核酸的新模式，并为未来的机制研究铺平道路。