Rad52-dependent recombination in response to replication stress

总结 基因组不稳定性是癌症的标志之一，是由复制压力驱动的。复制压力可以 由致癌基因激活、模板链损伤、核苷酸耗尽或由 复制叉进展的物理障碍，如非规范DNA结构或紧密 结合蛋白在停滞或折叠的复制叉处的同源重组（HR）对于 重新启动复制，但同时也可能是基因组不稳定性的另一个来源， 染色体重排最近的研究发现了Rad 52在促进DNA合成中的意想不到的作用。 在人类细胞中，它响应于复制应激而合成。虽然Rad 52对所有人力资源都至关重要， 芽殖酵母，它在哺乳动物细胞中的作用似乎很小，因为缺乏它的小鼠是可行的， Rad 52是BRCA 2缺陷细胞生存力所必需的这一发现， 被解释为介导Rad 51细丝组装的冗余。然而，新的研究 这表明Rad 52促进的DNA合成对复制应激的响应不依赖于Rad 51 提出了Rad 52新功能。因为Rad 52已经成为潜在的治疗靶点， 对于BRCA缺陷型肿瘤，了解其细胞功能非常重要。在这里，我们应用简易的 芽殖酵母的遗传学和新技术，以创造一个特定地点的复制叉停滞或崩溃 以确定在复制应激的情况下Rad 52依赖性重组的机制。在 该提案的第一个目标是，我们将使用Tus/Ter或Flp/FRT系统来诱导复制分叉停顿， 折叠，分别邻近敏感的报告分子，以检测分裂细胞中的HR。我们将确定 Tus/Ter和Flp/FRT刺激重组对Rad 51和Rad 52的要求，并鉴定 通过二维凝胶电泳检测重组中间体。汇聚分叉在 还将测试Tus/Ter块处的HR抑制。第二个目标涉及Rad 51的作用 稳定停滞的叉，新生链降解和Pol 32依赖的DNA合成， Tus/Ter诱导的重组。 !

SUMMARY Genomic instability, one of the hallmarks of cancer, is driven by replication stress. Replication stress can result from oncogene activation, damage to the template strands, depletion of nucleotides, or from physical impediments to progression of replication forks, such as non-canonical DNA structures or tightly bound proteins. Homologous recombination (HR) at stalled or collapsed replication forks is important to restart replication, but at the same time can be an additional source of genomic instability by promoting chromosome rearrangements. Recent studies identified an unexpected role for Rad52 in facilitating DNA synthesis in response to replication stress in human cells. Although Rad52 is essential for all HR in budding yeast, its role in mammalian cells had seemed minor because mice lacking it are viable and show only mild defects in HR. The finding that Rad52 is required for viability of BRCA2-deficient cells has been interpreted as redundancy for mediating Rad51 filament assembly. However, the new studies showing that Rad52-promoted DNA synthesis in response to replication stress is independent of Rad51 suggest a novel function for Rad52. Because Rad52 has emerged as potential therapeutic target for BRCA-deficient tumors, it is important to understand its cellular functions. Here we apply the facile genetics of budding yeast and new technologies to create a site-specific replication fork stall or collapse to identify the mechanism for Rad52 dependent recombination in the context of replication stress. In the first aim of the proposal, we will use Tus/Ter or Flp/FRT systems to induce a replication fork stall or collapse, respectively, adjacent to a sensitive reporter to detect HR in dividing cells. We will determine the requirements for Rad51 and Rad52 for Tus/Ter and Flp/FRT-stimulated recombination, and identify recombination intermediates by two-dimensional gel electrophoresis. The role of converging forks in suppressing HR at the Tus/Ter block will also be tested. The second aim addresses the role of Rad51 stabilization of stalled forks, nascent strand degradation and Pol32-dependent DNA synthesis for Tus/Ter-induced recombination. !