Regulation of DNA Damage and Repair in Heterochromatin

描述(申请人提供)：这个项目的目标是了解DNA修复机制如何有助于基因组异染色质区域内的基因组稳定性。重复DNA的复制、修复和重组可导致染色体重排和其他类型的基因组不稳定，这与癌症进展和其他人类疾病有关。利用果蝇模型系统，我们已经证明：1)异染色质建立和维持所需的染色质蛋白需要维持重复序列的稳定性，2)异染色质在照射后立即急剧膨胀，3)异染色质损伤的修复需要同源重组(HR)途径，以及4)HR修复的早期步骤发生在异染色质结构域内，但晚期事件只有在与重复序列相关的焦点移位到异染色质结构域之外后才发生。我们的工作假设是，重复序列的HR修复的潜在破坏性后果可以通过异染色质修复的异常空间和时间动态来避免，而异染色质修复是由异染色质成分介导的。我们建议使用果蝇动物和组织培养细胞来鉴定异色重复序列中调节DNA损伤反应的分子和机制。具体地说，我们将结合使用活细胞成像和固定细胞成像、突变分析、蛋白质生物化学和基因组方法来确定异染色质扩张、修复焦点动态和同源重组是如何在重复DNA中调节的，以及这些事件如何有助于基因组的稳定性。这些研究的结果将大大提高我们对异染色质环境如何确保重复序列的稳定性的理解，这在人类疾病的诊断和治疗中具有重要应用。

DESCRIPTION (provided by applicant): The goal of this project is to understand how DNA repair mechanisms contribute to genome stability within the heterochromatic regions of the genome. Replication, repair and recombination of repeated DNAs can result in chromosome rearrangements and other types of genome instability, which are associated with cancer progression and other human diseases. Using the Drosophila model system, we have shown that 1) chromatin proteins required for heterochromatin establishment and maintenance are required to preserve the stability of repeated sequences, 2) heterochromatin undergoes a dramatic expansion immediately after irradiation, 3) repair of damage in heterochromatin requires the homologous recombination (HR) pathway, and 4) early steps in HR repair occur within the heterochromatin domain, but late events only occur after foci associated with repeated sequences translocate outside the heterochromatin domain. Our working hypothesis is that the potentially damaging consequences of HR repair of repeated sequences are avoided by the unusual spatial and temporal dynamics of heterochromatin repair, which are mediated by heterochromatin components. We propose to use Drosophila animals and tissue culture cells to identify the molecules and mechanisms that regulate the response to DNA damage in heterochromatic, repeated sequences. Specifically, we will use a combination of live and fixed cell imaging, mutant analysis, protein biochemistry, and genomic approaches to determine how heterochromatin expansion, repair foci dynamics, and homologous recombination are regulated at repeated DNAs, and how these events contribute to genome stability. The results of these studies will greatly improve our understanding of how the heterochromatin environment ensures the stability of repeated sequences, which has important applications to the diagnosis and treatment of human diseases.