Molecular mechanisms of pathway choice in DNA double strand break repair

项目摘要 DNA双链断裂(DSB)是一种特别有毒的DNA损伤形式。即使是一个DSB也可能导致 细胞死亡。我们的细胞使用许多复杂的修复途径来修复DSB。对于大多数休息时间来说， 在我们的细胞中发生的一种称为非同源末端连接(NHEJ)的途径被用来有效地将 断了的线又聚在一起了。或者，DNA末端的一条链被“咬回”，这个过程被称为 切除会产生单链DNA悬垂。多条路径作用于这些悬挑 包括同源重组(HR)和微同源介导的末端连接(MMEJ)。人力资源是一个错综复杂的 当MMEJ是诱变剂时，使用姐妹染色单体以高保真的方式指导修复的机制 其机制与NHEJ更相似的途径。正确选择这些修复路径是至关重要的 对于人类健康，因为滥用与可能导致癌症的严重染色体变化相关。经常 癌细胞缺乏DNA修复途径，这使它们能够迅速获得不正常的特征 与健康细胞有关。这些修复缺陷也使它们容易受到靶向癌症的攻击。 治疗。 这项提议试图从分子水平上更好地理解细胞如何在这两者之间进行选择。 不同的DSB修复途径。在细胞中的实验已经确定了一些在此过程中起作用的蛋白质。 分子决策过程，但我们缺乏对这些蛋白质如何协同工作的了解。 传统上，这种知识是通过纯化单个蛋白质并将它们组合在一起来获得的 重新构建一个生物过程。然而，考虑到蛋白质的绝对数量，目前无法维持 这样一种方法。我们最近验证了一种从非洲爪蛙卵中提取的无细胞提取物 LEVIS系统概括了DSB途径的选择。将这一生理生化系统与 在单分子水平上研究DNA DSB修复蛋白动力学的强大成像手段， 我们将阐明DSB修复途径选择的分子基础。具体地说，我们将努力澄清 抗DNA末端切除的NHEJ因子Ku被从DNA末端去除(目标1)； MMEJ聚合酶Polθ在部分切除的悬突上与远距离切除竞争(目标2)。最后， 我们将阐明多功能POLθ是如何利用其不同的酶活性来突触DNA端和 搜索微同源性(目标3)。我们的研究将揭示DSB修复途径的重要新见解 选拔和规范。这些洞察力可能会使人们能够以新的方式改变这些修复之间的平衡 在基因编辑或治疗缺乏DSB修复的癌症中可能有应用的途径。

Project Summary DNA double strand breaks (DSBs) are a particularly toxic form of DNA damage. Even a single DSB can lead to cell death. Our cells use a number of intricate repair pathways to repair DSBs. For the majority of breaks that occur in our cells a pathway known as non-homologous end joining (NHEJ) is used to effectively “glue” the broken strands back together. Alternatively, a strand at each DNA end is “chewed back” in a process known as resection which generates single-stranded DNA overhangs. Multiple pathways act on these overhangs including homologous recombination (HR) and microhomology mediated end joining (MMEJ). HR is an intricate mechanism which uses a sister chromatid to direct repair in a high-fidelity manner while MMEJ is a mutagenic pathway whose mechanism more closely resembles NHEJ. Proper selection of these repair pathways is critical for human health as misuse is correlated with gross chromosomal changes that can result in cancer. Often cancer cells are deficient in DNA repair pathways which allows them to rapidly acquire traits not normally associated with healthy cells. These repair deficiencies also make them vulnerable to targeted cancer therapies. This proposal seeks to develop a better molecular understanding of how cells choose between these different DSB repair pathways. Experiments in cells have identified a number of proteins that play a role in this molecular decision-making process, but we lack an understanding of how these proteins work together. Traditionally, such knowledge is gained by purifying individual proteins and combining them together to reconstitute a biological process. However, given the sheer number of proteins, it is currently untenable to take such an approach. We have recently validated a cell-free extract made from the eggs of the frog Xenopus laevis as system that recapitulates DSB pathway choice. Combining this physiological biochemical system with powerful imaging approaches to study the dynamics of DNA DSB repair proteins at the single-molecule level, we will elucidate the molecular basis of DSB repair pathway choice. Specifically, we will work to clarify how the NHEJ factor Ku, which antagonizes DNA end resection, is removed from DNA ends (Aim 1); and how the MMEJ polymerase Pol θ competes with long-range resection on partially resected overhangs (Aim 2). Finally, we will elucidate how the multi-functional Pol θ uses its diverse enzymatic activities to synapse DNA ends and search for microhomology (Aim 3). Our studies will reveal significant new insights into DSB repair pathway selection and regulation. These insights may enable novel ways to alter the balance between these repair pathways which could have applications in gene editing or in therapies for cancers deficient in DSB repair.