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

在细胞分裂之前，DNA复制复制基因组，因此所有染色体的副本可以传递给两个初生子细胞中的每一个。为了实现这一点，父母的染色体在复制起始处解开，形成DNA复制叉子。这些是复制型DNA聚合酶催化DNA合成的位置。当复制叉沿着染色体跟踪时，它们通常会因一系列障碍而停滞不前，这些障碍包括聚合酶阻断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条件，进一步增强了复制压力。其结果是，复制叉会停滞或断裂，危及基因组复制，并使细胞暴露在染色体分离问题、染色体断裂和总染色体不稳定中。为了抵消这些对基因组稳定性的威胁，细胞进化了复制分叉恢复机制。值得注意的是，受到干扰的叉子恢复与包括原始侏儒症和肿瘤发生在内的人类疾病有关。相反，由于癌细胞产生固有的复制压力，靶向复制分叉恢复通路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重要的科学意义和生物医学意义。细胞在停滞的复制叉重新启动DNA复制的方式之一依赖于同源重组。这需要解离新生的DNA链，它经历了亲本染色体的入侵，形成了所谓的置换环(D-loop)。依赖于聚合酶POL32亚单位(POLD3)的D-loop DNA合成成为新的复制模式。这个过程通常是有益的，有助于细胞克服即使是顽固的复制障碍。然而，D-loop DNA合成容易出错和不稳定，会导致异位重组和染色体重排。因此，一个关键的问题是细胞如何控制重组依赖的复制叉恢复，以平衡复制完成的好处和该途径对基因组稳定带来的风险。我们最近报道，疾病相关的DNA2核酸酶/解旋酶是停滞的复制叉的关键处理因素，对完成染色体复制是严格要求的。此外，我们认为DNA2的作用限制了重组依赖的分叉重新启动的使用，并且在没有DNA2的情况下过度重组对细胞是有害的。这种重组的“把关人”模型为DNA2跨生物体的本质提供了一个新的理论基础，但仍有待检验。与这一模型一致，我们发现了新的Pol32功能分离突变，这些突变特异性地使D-loop DNA合成失效，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来解决关键问题，即细胞如何在停滞的复制分叉控制同源重组，并在DNA复制的重新开始过程中实现恢复途径的适当平衡。我们将直接在复制停滞的特定基因组位置测试DNA2守门人假说。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合成的难以捉摸的需求。第三，我们将研究细胞如何通过我们确定的翻译后修饰来指示Pol32作为变阻器来控制重组介导的复制的水平。这一工作计划将提供前所未有的机制洞察DNA2和POL32(POLD3)在控制复制分叉恢复和抵消复制压力中的作用。这些结论将有助于将DNA2和POLD3疾病与塞克尔综合征、线粒体肌病和癌症的联系合理化。

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.