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.

这项工作的总体目标是提高我们对人类生育能力分子基础的理解。最近发现，复制和修复 DNA 分子的蛋白质与生育特征（包括绝经年龄和卵巢衰老）之间存在密切联系。这种关系被认为反映了 DNA 修复机制在精子和卵细胞的生成以及它们所包含的遗传信息的维护中所发挥的重要作用。该项目专注于一种称为 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，这种蛋白质的突变与早发的更年期密切相关，但我们完全没有关于 HELB 中的这些分子缺陷如何影响其细胞功能的信息。这在一定程度上反映了 HELB 是一种研究较少的蛋白质这一事实；尽管多项研究表明 HELB 有助于 DNA 维持，但其在这些途径中的确切作用仍不清楚。此外，我们不了解 HELB 蛋白的结构，也不了解它与其他复制和修复因子形成的复合物。最近，我们发现了 HELB 功能的一条重要线索，表明它可以作为 DNA 马达蛋白从单链 DNA (ssDNA) 中剥离 RPA。 RPA 是一种与 ssDNA 快速紧密结合的蛋白质，可保护其免遭降解，形成的 RPA-ssDNA 细丝是许多复制和修复过程中的关键中间体。然而，随后从这些中间体中去除 RPA 对于完成复制和修复的下游因子的结合至关重要。因此，我们认为 RPA 重塑是 HELB 在与其相关的所有不同途径中的潜在作用。值得注意的是，结构模型研究现在表明，与早发更年期相关的 HELB 缺陷会影响其与 RPA 相互作用的能力，这一假设是我们研究提案的基础。在这项工作中，我们将：（1）确定 HELB 的架构RPA 复合体揭示了 HELB 和 RPA 之间形成的接口的原子细节。通过这种方式，我们将测试 HELB 中的缺陷如何影响 HELB 与 RPA 的结合。 (2) 确定 HELB-RPA 界面以及我们认为破坏该界面的 HELB 突变对于 RPA 丝上的 HELB 活性和 HELB 依赖性 DNA 修复的重要性。我们的项目将从分子水平上对 HELB 的结构和功能产生新的见解，以及 HELB 的遗传缺陷、HELB-RPA 界面的结构扰动、功能失调的 RPA 丝重塑、遗传不稳定性和（最终）人类生育能力之间的联系问题。这项基础研究将增进我们对人类生殖性能的更广泛理解，并将支持进一步的转化研究以解决不孕症问题。