The interaction of HELB with RPA and its role in human fertility

HELB 与 RPA 的相互作用及其在人类生育力中的作用

• 批准号: MR/Y012070/1
• 负责人: Mark Dillingham
• 金额: $ 67.85万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FY012070%2F1
• 关键词: interaction; HELB; RPA; its; role

The overarching objective of this work is to improve our understanding of the molecular basis for human fertility. It has recently become apparent that there are strong links between proteins which replicate and repair DNA molecules and fertility traits including age at menopause and ovarian ageing. This relationship is thought to reflect the important role played by the DNA repair machinery in the generation of sperm and egg cells and the maintenance of the hereditary information they contain. This project focusses on one particular DNA replication and repair factor called DNA helicase B (HELB). Geneticists have shown that mutation of this protein is strongly implicated in early onset menopause but we have no information at all about how these molecular defects in HELB affect its cellular functions. This partly reflects the fact that HELB is a poorly-studied protein; although several studies have shown that HELB contributes to DNA maintenance its precise roles in these pathways remain undefined. Furthermore, we have no understanding of the architecture of the HELB protein, nor of the complexes it forms with other replication and repair factors. We recently found one important clue to the function of HELB when we showed that it acts as a DNA motor protein to strip RPA from single-stranded DNA (ssDNA). RPA is a protein which binds rapidly and tightly to ssDNA to protect it from degradation, and the RPA-ssDNA filaments that are formed are key intermediates in many replication and repair processes. However, the subsequent removal of RPA from these intermediates is essential for the binding of downstream factors which complete replication and repair. Consequently, we have suggested that RPA remodeling is the underlying role of HELB in all of the varied pathways it has been associated with. Remarkably, structural modelling studies now suggest that the HELB defects associated with early onset menopause affect its ability to interact with RPA and this hypothesis is the basis for our research proposal.In this work we will:(1) Determine the architecture of the HELB-RPA complex to unveil the atomic details of the interface formed between HELB and RPA. In this way we will test how defects in HELB affect the binding of HELB to RPA. (2) Determine the importance of the HELB-RPA interface, and of the HELB mutations that we think disrupt it, for HELB activity on RPA filaments and HELB-dependent DNA repair. Our project will generate novel insights into the structure and function of HELB at the molecular level, and also the links between genetic defects in HELB, structural perturbations of the HELB-RPA interface, dysfunctional RPA filament remodeling, genetic instability and (ultimately) human fertility problems. This basic research will improve our broader understanding of human reproductive performance and will support further translational research to address infertility.

这项工作的总体目的是提高我们对人类生育分子基础的理解。最近显而易见的是，复制和修复DNA分子的蛋白质之间存在牢固的联系，包括更年期和卵巢衰老的年龄，包括年龄。人们认为这种关系反映了DNA修复机械在生成精子和卵细胞中所起的重要作用，以及维持所包含的遗传信息。该项目的重点是一种称为DNA解旋酶B（HELB）的特定DNA复制和修复因子。遗传学家已经表明，该蛋白的突变与早期发作很大，但我们根本没有有关这些分子缺陷如何影响其细胞功能的信息。这部分反映了HELB是一种研究不佳的蛋白质。尽管几项研究表明，HELB有助于DNA维持其在这些途径中的精确作用，但仍未定义。此外，我们对HELB蛋白的结构没有任何了解，也不了解其与其他复制和修复因子形成的复合物。最近，当我们表明它是从单链DNA（SSDNA）剥离RPA的DNA运动蛋白时，我们发现了HELB功能的一个重要线索。 RPA是一种蛋白质，可快速和紧密结合ssDNA以保护其免受降解，而形成的RPA-SSDNA丝是许多复制和修复过程中的关键中间体。但是，随后从这些中间体中删除RPA对于完全复制和修复的下游因子的结合至关重要。因此，我们建议RPA重塑是HELB在与之相关的所有不同途径中的基本作用。值得注意的是，结构建模研究现在表明，与早期发作更年期相关的HELB缺陷会影响其与RPA相互作用的能力，而该假设是我们的研究建议的基础。在这项工作中，我们将：（1）确定Helb-RPA复合体的结构，以揭示HELB和RPA之间界面的原子细节。通过这种方式，我们将测试HELB缺陷如何影响HELB与RPA的结合。 （2）确定HELB-RPA界面的重要性以及我们认为破坏其的HELB突变的重要性。我们的项目将产生对HELB在分子水平上的结构和功能的新见解，以及HELB中的遗传缺陷，HELB-RPA界面的结构扰动，功能障碍的RPA细丝重塑，遗传不稳定性，遗传不稳定性和（最终）人类生育问题之间的联系。这项基础研究将提高我们对人类生殖绩效的更广泛的理解，并将支持进一步的转化研究以解决不孕症。