Astbury Centre for Structural Molecular Biology, School of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CambridgeCB2 1GA, UK.
Nucleic Acids Res. 2024 Nov 11;52(20):12405-12421. doi: 10.1093/nar/gkae831.
DNA damage that obstructs the replication machinery poses a significant threat to genome stability. Replication-coupled repair mechanisms safeguard stalled replication forks by coordinating proteins involved in the DNA damage response (DDR) and replication. SLF1 (SMC5-SMC6 complex localization factor 1) is crucial for facilitating the recruitment of the SMC5/6 complex to damage sites through interactions with SLF2, RAD18, and nucleosomes. However, the structural mechanisms of SLF1's interactions are unclear. In this study, we determined the crystal structure of SLF1's ankyrin repeat domain bound to an unmethylated histone H4 tail, illustrating how SLF1 reads nascent nucleosomes. Using structure-based mutagenesis, we confirmed a phosphorylation-dependent interaction necessary for a stable complex between SLF1's tandem BRCA1 C-Terminal domain (tBRCT) and the phosphorylated C-terminal region (S442 and S444) of RAD18. We validated a functional role of conserved phosphate-binding residues in SLF1, and hydrophobic residues in RAD18 that are adjacent to phosphorylation sites, both of which contribute to the strong interaction. Interestingly, we discovered a DNA-binding property of this RAD18-binding interface, providing an additional domain of SLF1 to enhance binding to nucleosomes. Our results provide critical structural insights into SLF1's interactions with post-replicative chromatin and phosphorylation-dependent DDR signalling, enhancing our understanding of SMC5/6 recruitment and/or activity during replication-coupled DNA repair.
阻碍复制机制的 DNA 损伤对基因组稳定性构成重大威胁。复制偶联修复机制通过协调参与 DNA 损伤反应 (DDR) 和复制的蛋白质来保护停滞的复制叉。SLF1（SMC5-SMC6 复合物定位因子 1）对于通过与 SLF2、RAD18 和核小体相互作用促进 SMC5/6 复合物招募到损伤部位至关重要。然而，SLF1 相互作用的结构机制尚不清楚。在这项研究中，我们确定了 SLF1 的锚蛋白重复结构域与未甲基化组蛋白 H4 尾巴结合的晶体结构，说明了 SLF1 如何读取新生核小体。使用基于结构的诱变，我们证实了 SLF1 的串联 BRCA1 C 末端结构域（tBRCT）和 RAD18 的磷酸化 C 末端区域（S442 和 S444）之间的一个稳定复合物所必需的磷酸化依赖性相互作用。我们验证了 SLF1 中的保守磷酸结合残基和 RAD18 中紧邻磷酸化位点的疏水性残基的功能作用，这两者都有助于这种强相互作用。有趣的是，我们发现了这个 RAD18 结合界面的 DNA 结合特性，为 SLF1 提供了一个额外的结构域来增强与核小体的结合。我们的研究结果为 SLF1 与复制后染色质的相互作用和磷酸化依赖性 DDR 信号转导提供了关键的结构见解，增强了我们对 SMC5/6 在复制偶联 DNA 修复过程中的募集和/或活性的理解。
