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

这项工作的首要目标是提高我们对人类生育的分子基础的理解。最近发现，复制和修复DNA分子的蛋白质与包括绝经年龄和卵巢老化在内的生育特征之间存在着强烈的联系。这种关系被认为反映了DNA修复机制在精子和卵细胞的产生以及它们所包含的遗传信息的维护中发挥的重要作用。这个项目专注于一种特殊的DNA复制和修复因子，称为DNA解旋酶B(HELB)。遗传学家已经证明，这种蛋白的突变与早期绝经密切相关，但我们完全不知道Helb的这些分子缺陷如何影响其细胞功能。这在一定程度上反映了这样一个事实，即Helb是一种研究较少的蛋白质；尽管几项研究表明，Helb有助于DNA的维持，但它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体。最近，我们发现了Helb功能的一条重要线索，因为我们证明了它作为DNA马达蛋白从单链DNA(SsDNA)中剥离RPA。RPA是一种能快速、紧密地与单链DNA结合以防止其降解的蛋白质，所形成的RPA-单链DNA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移除RPA对于结合完成复制和修复的下游因子是必不可少的。因此，我们认为RPA重塑是HELB在与之相关的所有不同途径中的潜在作用。值得注意的是，结构建模研究现在表明，与早期绝经相关的Helb缺陷影响其与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-RPA复合体的结构，以揭示Helb和RPA之间形成的界面的原子细节。通过这种方式，我们将测试Helb中的缺陷如何影响Helb到RPA的绑定。(2)确定Helb-RPA界面以及我们认为破坏它的Helb突变对RPA细丝上的Helb活性和Helb依赖的DNA修复的重要性。我们的项目将在分子水平上对HELB的结构和功能产生新的见解，以及HELB中的遗传缺陷、HELB-RPA界面的结构扰动、功能失调的RPA细丝重塑、遗传不稳定性以及(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能力的更广泛理解，并将支持进一步的翻译研究，以解决不孕不育问题。

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.