Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560012, India.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560012, India.
Nucleic Acids Res. 2024 Feb 9;52(3):1450-1470. doi: 10.1093/nar/gkad1208.
The regulator of telomere elongation helicase 1 (RTEL1) plays roles in telomere DNA maintenance, DNA repair, and genome stability by dismantling D-loops and unwinding G-quadruplex structures. RTEL1 comprises a helicase domain, two tandem harmonin homology domains 1&2 (HHD1 and HHD2), and a Zn2+-binding RING domain. In vitro D-loop disassembly by RTEL1 is enhanced in the presence of replication protein A (RPA). However, the mechanism of RTEL1 recruitment at non-telomeric D-loops remains unknown. In this study, we have unravelled a direct physical interaction between RTEL1 and RPA. Under DNA damage conditions, we showed that RTEL1 and RPA colocalise in the cell. Coimmunoprecipitation showed that RTEL1 and RPA interact, and the deletion of HHDs of RTEL1 significantly reduced this interaction. NMR chemical shift perturbations (CSPs) showed that RPA uses its 32C domain to interact with the HHD2 of RTEL1. Interestingly, HHD2 also interacted with DNA in the in vitro experiments. HHD2 structure was determined using X-ray crystallography, and NMR CSPs mapping revealed that both RPA 32C and DNA competitively bind to HHD2 on an overlapping surface. These results establish novel roles of accessory HHDs in RTEL1's functions and provide mechanistic insights into the RPA-mediated recruitment of RTEL1 to DNA repair sites.
端粒延伸螺旋酶 1（RTEL1）调节剂通过拆开 D 环和解开 G-四链体结构，在端粒 DNA 维持、DNA 修复和基因组稳定性方面发挥作用。RTEL1 包含一个螺旋酶结构域、两个串联的 harmonin 同源结构域 1&2（HHD1 和 HHD2）和一个 Zn2+结合的 RING 结构域。体外实验表明，RTEL1 对 D 环的解旋作用在复制蛋白 A（RPA）存在时增强。然而，RTEL1 在非端粒 D 环上的募集机制仍然未知。在这项研究中，我们揭示了 RTEL1 和 RPA 之间的直接物理相互作用。在 DNA 损伤条件下，我们表明 RTEL1 和 RPA 在细胞中共定位。共免疫沉淀表明 RTEL1 和 RPA 相互作用，并且 RTEL1 的 HHD 缺失显著降低了这种相互作用。NMR 化学位移扰动（CSP）表明 RPA 使用其 32C 结构域与 RTEL1 的 HHD2 相互作用。有趣的是，HHD2 也在体外实验中与 DNA 相互作用。使用 X 射线晶体学确定了 HHD2 的结构，并且 NMR CSP 映射表明 RPA 32C 和 DNA 竞争性地在重叠表面上与 HHD2 结合。这些结果确立了辅助 HHD 在 RTEL1 功能中的新作用，并为 RPA 介导的 RTEL1 募集到 DNA 修复位点提供了机制见解。
