Investigating the Mechanisms Controlling Homologous Recombination-Dependent DNA Replication Fork Recovery in Response to Replication Stress.

研究控制同源重组依赖的 DNA 复制叉恢复以应对复制压力的机制。

• 批准号: BB/W008505/1
• 负责人: Ulrich Rass
• 金额: $ 51.46万
• 依托单位: University of Sussex
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW008505%2F1
• 关键词: Investigating; Mechanisms; Controlling; Homologous; Recombination; Investigating; Mechanisms; Controlling; Homologous; Recombination

Before cells divide, DNA replication duplicates the genome, so that a copy of all chromosomes may transmitted to each of two incipient daughter cells. To achieve this, the parental chromosomes become unwound at origins of replication, forming DNA replication forks. These are the sites where replicative DNA polymerases catalyse DNA synthesis. As replication forks track along the chromosomes, they are routinely stalled by a range of obstacles including polymerase-blocking DNA lesions, DNA secondary structures, DNA-binding proteins, and DNA-RNA hybrids; this is referred to as DNA replication stress. Unfavourable replication conditions, such as those found in dysregulated cancer cells, further enhance replication stress. As a consequence, replication forks stall or break, jeopardising genome duplication, and exposing cells to chromosome segregation problems, chromosome breakage, and gross chromosomal instability. To offset these threats to genome stability, cells have evolved replication fork recovery mechanisms. Notably, perturbed fork recovery has been linked to human diseases including primordial dwarfism and tumorigenesis. Conversely, because cancer cells generate intrinsic replication stress, targeting replication fork recovery pathways has emerged as a potential anti-cancer strategy. Thus, understanding the mechanisms and regulation of replication fork recovery is of great scientific interest and biomedical importance.One of the ways in which cells reboot DNA replication at stalled replication forks is dependent upon homologous recombination. This requires the dissociation of a nascent DNA strand, which undergoes invasion of the parental chromosome to from a so-called displacement loop (D-loop). D-loop DNA synthesis, which uniquely depends upon the polymerase subunit POL32 (POLD3), then becomes the new mode of replication. This process is generally beneficial, helping cells to overcome even tenacious replication obstacles. However, D-loop DNA synthesis is error prone and unstable, which can cause ectopic recombination and chromosome rearrangements. A key question, therefore, is how cells control recombination-dependent replication fork recovery to balance the benefits to replication completion with the risks the pathway poses to genome stability.We have recently reported that the disease-associated DNA2 nuclease/helicase is a critical processing factor at stalled replication forks, strictly required for the completion of chromosome replication. Furthermore, we suggested that the actions of DNA2 limit the use of recombination-dependent fork restart, and that excessive recombination in the absence of DNA2 is toxic for cells. This recombination "gatekeeper" model has provided a new rationale for the essential nature of DNA2 across organisms, but remains to be tested. Consistent with this model, we have identified new Pol32 separation-of-function mutations that specifically disable D-loop DNA synthesis, and concomitantly rescue the viability of DNA2-defective cells. Here, we propose to exploit these findings to unlock key questions of how cells control homologous recombination at stalled replication forks and implement the appropriate balance of recovery pathways in the restart of DNA replication. We will test the DNA2 gatekeeper hypothesis directly at a defined genomic site of replication stalling. Secondly, we will leverage the interactions between DNA2 and POL32 to reveal the elusive requirement of POL32 for D-loop DNA synthesis. And thirdly, we will examine how cells instruct Pol32 by post-translational modifications we identified, to act as a rheostat controlling the levels of recombination-mediated replication.This work programme will provide unprecedented mechanistic insight into the roles of DNA2 and POL32 (POLD3) in controlling replication fork recovery and offsetting replication stress. The conclusions will help rationalize DNA2 and POLD3 disease links with Seckel syndrome, mitochondrial myopathy, and cancer.

在细胞分裂之前，DNA复制重复了基因组，因此所有染色体的副本都可以传递到两个初期的子细胞中的每个染色体。为了实现这一目标，父母染色体在复制的起源中变得不合适，形成了DNA复制叉。这些是复制性DNA聚合酶催化DNA合成的位点。随着复制叉沿染色体的轨迹，它们通常会被包括聚合酶阻断DNA病变，DNA二级结构，DNA结合蛋白和DNA-RNA杂种在内的一系列障碍物而停滞。这称为DNA复制应力。不利的复制条件，例如在失调的癌细胞中发现的复制条件，进一步增强了复制应力。结果，复制叉档或破裂，危害基因组重复，并将细胞暴露于染色体分离问题，染色体断裂和染色体不稳定。为了抵消对基因组稳定性的这些威胁，细胞已经发展出复制叉恢复机制。值得注意的是，受扰动的叉恢复与包括原始矮人和肿瘤发生在内的人类疾病有关。相反，由于癌细胞会产生内在的复制应力，因此靶向复制叉恢复途径已成为潜在的抗癌策略。因此，理解复制叉恢复的机制和调节是非常科学的兴趣和生物医学重要性。一种细胞在停滞的复制叉时重新启动DNA复制的方式的一种取决于同源重组。这需要新生DNA链的解离，该链从所谓的位移环（D-Loop）中侵袭了父母染色体。 D-Loop DNA合成，它独特地取决于聚合酶亚基POL32（POLD3），然后成为新的复制方式。这个过程通常是有益的，可以帮助细胞克服甚至顽强的复制障碍。但是，D-Loop DNA合成是易误和不稳定的，这可能导致异位重组和染色体重排。因此，一个关键问题是细胞如何控制重组依赖性复制叉恢复以平衡复制完成的好处与途径对基因组稳定性构成的风险。我们最近报道说，与疾病相关的DNA2核酸酶/解旋酶是严格的复制叉的关键处理因子，严格要求完成Chromosomosososomesomemososomesomemosomesomemosome的复制。此外，我们建议DNA2的作用限制了使用重组依赖性叉的使用，并且在没有DNA2的情况下过度重组对细胞有毒。这种重组“守门人”模型为跨生物体DNA2的基本性质提供了新的理由，但仍有待测试。与该模型一致，我们已经确定了新的POL32分离功能突变，这些突变专门禁用D-Loop DNA合成，并同时挽救了DNA2缺陷细胞的生存能力。在这里，我们建议利用这些发现，以解锁细胞如何控制停滞的复制叉中同源重组的关键问题，并在DNA复制重新启动中实现恢复途径的适当平衡。我们将直接在定义的复制基因组位点直接测试DNA2网守假设。其次，我们将利用DNA2和POL32之间的相互作用来揭示POL32对D-Loop DNA合成的难以捉摸的要求。第三，我们将检查细胞如何通过我们确定的翻译后修饰来指导POL32，以充当控制重组介导的复制水平的恒河体。这项工作计划将提供对DNA2和POL32（POLD3）在控制复制复制Fork Rouseconfork Recousek Recousek Recouseke Recousepection Repeplate Repeplatitation Repeplateptions的作用中的前所未有的机械洞察力。这些结论将有助于将DNA2和POLD3疾病与SECKEL综合征，线粒体肌病和癌症进行合理化。