Investigating DNA double-strand break repair mechanisms in mammalian cells

主要调查者：吴晓华 项目摘要 总染色体重排(GCR)是癌细胞的一个标志，而 遗传性疾病中结构变异形成的原因。DNA双链断裂(DSB) 是导致GCR的主要来源，但关于DSB修复过程如何导致 癌症和其他遗传疾病的基因组不稳定性仍然难以捉摸。 微同源物普遍存在于癌症和其他疾病的染色体断裂连接处 人类疾病，这表明微同源介导的末端连接(MMEJ)是主要的 调停GCR的玩家。重复序列丰富，约占 人类基因组，这提出了一个问题，即重复序列之间的重组如何 序列被抑制，以防止基因组不稳定。在这项建议中，我们的目标是 研究MMEJ和重复序列介导的重组的机制 哺乳动物细胞。 我们已经建立了多个基于EGFP的DSB修复报告系统，其中 使我们能够研究哺乳动物细胞中不同的DSB修复途径。通过使用新的 建立了MMEJ记者，我们在MMEJ途径中发现了新的参与者。我们会 调查这些MMEJ玩家如何调节MMEJ以平衡其使用和诱变性 后果。我们还将研究MMEJ在修复复制中的生物学作用- 相关的DSB以及在应对复制和致癌压力方面的作用，并解决如何 MMEJ有助于基因组的不稳定性和致癌过程。此外，我们还将 确定重复序列介导的重组在导致缺失和 易位，并研究异源双链排斥在防止重组中的作用 在不同的重复之间。这些研究将产生急需的新知识来填补 我们对DSB修复机制和基因组成因的认识差距 与致癌过程和其他遗传性疾病有关的不稳定性。

Principal Investigator: Wu, Xiaohua Project Summary Gross chromosomal rearrangement (GCR) is a hallmark of cancer cells and the cause of structural variant formation in genetic disorders. DNA double-strand breaks (DSBs) are a major source to induce GCR, but the details on how the DSB repair process leads to genome instability in cancers and other genetic disorders remains elusive. Microhomologies are prevalent at chromosomal break junctions in cancer and other human diseases, suggesting that microhomology-mediated end joining (MMEJ) is one major player to mediate GCRs. Repetitive sequences are abundant, constituting about half of the human genome, which raises the question of how recombination between repetitive sequences is suppressed to prevent genome instability. In this proposal, we aim to investigate the mechanisms underlying MMEJ and repeat-mediated recombination in mammalian cells. We have established multiple EGFP-based DSB repair reporter systems, which enable us to study different DSB repair pathways in mammalian cells. By using newly established MMEJ reporters, we identified new players in the MMEJ pathway. We will investigate how these MMEJ players regulate MMEJ to balance its usage and its mutagenic consequences. We will also study the biological role of MMEJ in repairing replication- associated DSBs and in coping with replication and oncogenic stress, and address how MMEJ contributes to genome instability and the oncogenic process. Additionally, we will determine the mechanisms of repeat-mediated recombination in causing deletions and translocations, and study the role of heteroduplex rejection in preventing recombination among divergent repeats. These studies will generate much-needed new knowledge to fill the gap in our understanding of DSB repair mechanisms and the cause of genome instability that is associated with the oncogenic process and other genetic disorders.