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

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复制复制基因组，因此所有染色体的拷贝可以传递到两个初始子细胞中的每一个。为了实现这一点，亲本染色体在复制起点处展开，形成DNA复制叉。这些是复制型DNA聚合酶催化DNA合成的位点。当复制叉沿着染色体追踪时，它们通常会受到一系列障碍的阻碍，包括聚合酶阻断的DNA损伤、DNA二级结构、DNA结合蛋白和DNA-RNA杂交体;这被称为DNA复制应激。不利的复制条件，如在失调的癌细胞中发现的那些，进一步增强复制应激。结果，复制叉停滞或断裂，危及基因组复制，并使细胞暴露于染色体分离问题、染色体断裂和总染色体不稳定性。为了抵消这些对基因组稳定性的威胁，细胞已经进化出复制叉恢复机制。值得注意的是，干扰叉子恢复与人类疾病有关，包括原始侏儒症和肿瘤发生。相反，由于癌细胞产生内在复制应激，靶向复制叉恢复途径已成为一种潜在的抗癌策略。因此，了解复制叉恢复的机制和调控具有极大的科学兴趣和生物医学意义。细胞在停滞的复制叉处重新启动DNA复制的方式之一依赖于同源重组。这需要新生DNA链的解离，其经历亲本染色体的侵入以形成所谓的置换环（D环）。D-loop DNA合成，这唯一依赖于聚合酶亚基POL 32（POLD 3），然后成为新的复制模式。这个过程通常是有益的，帮助细胞克服甚至是顽强的复制障碍。然而，D-环DNA合成容易出错且不稳定，这可引起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造成的风险。我们最近报道，疾病相关的DNA 2核酸酶/解旋酶是停滞复制叉的关键加工因子，严格要求完成染色体复制。此外，我们认为DNA 2的作用限制了重组依赖性叉重启动的使用，并且在DNA 2不存在的情况下过度重组对细胞是有毒的。这种重组“看门人”模型为DNA 2跨生物体的基本性质提供了新的理论基础，但仍有待检验。与此模型一致，我们已经确定了新的Pol 32分离功能突变，特异性地禁用D-环DNA合成，并伴随着拯救DNA 2缺陷细胞的活力。在这里，我们建议利用这些发现来解开细胞如何在停滞的复制叉控制同源重组的关键问题，并在DNA复制的重新启动中实现恢复途径的适当平衡。我们将直接在复制停滞的确定基因组位点测试DNA 2看门人假设。其次，我们将利用DNA 2和POL 32之间的相互作用来揭示POL 32对D环DNA合成的难以捉摸的要求。第三，我们将研究细胞如何通过我们鉴定的翻译后修饰来指导Pol 32，作为控制重组介导的复制水平的变阻器。这项工作计划将为DNA 2和POL 32（POLD 3）在控制复制叉恢复和抵消复制应激中的作用提供前所未有的机制见解。这些结论将有助于合理化DNA 2和POLD 3疾病与Seckel综合征，线粒体肌病和癌症的联系。