Novel pathways that regulate DNA double-strand break repair events in mammalian cells

摘要摘要 我们研究的总体愿景是发现组蛋白和非组蛋白 DNA上的蛋白质，即染色质，调节基因组过程和衰老。特别是，我们努力将 不同的领域，如染色质在基因组稳定性中的作用以及染色质在衰老中的作用。使用 生物化学、结构生物学、芽殖酵母的分子遗传学、组织培养和 全基因组的方法，我们发现染色质在没有 不仅基因表达和DNA复制，而且在DNA双链断裂修复过程中也是如此。我们有 揭示了这些事件的机制基础及其对这些基因组过程的关键影响。在更多方面 近年来，我们已经将我们解决的问题扩展到染色质之外--例如，揭示 衰老的新机制基础和发现延长寿命的新方法。同样，受我们的启发， 最近发现染色质结构将DNA双链断裂的过程减少为单链 DNA(称为DNA末端切除)，我们设计了创新的CRISPR/Cas9 gRNA文库筛选 识别在DNA双链修复过程中调节DNA末端切除的新活动的方法。 人体内大多数细胞处于G0/G1期，过量的DNA最终被切除是至关重要的 在这些细胞中不会发生。如果发生这种情况，它将通过唯一用于治疗的途径阻止DNA修复 修复G0/G1期细胞DNA双链断裂，即非同源末端连接(NHEJ) 会导致随后的同源基因介导的修复中的移位和缺失。事实上，这一程度 在使用NHEJ或同种异体之间的选择中，DNA末端切除是关键的决策点 修复DNA双链断裂的重组(HR)途径。我们建议，机制必须是 采取措施限制在G0/G1期细胞中过量DNA切除，以防止HR，同时允许足够的DNA 结束未连接的DNA末端的处理，以允许NHEJ介导的修复。这些蛋白质和信号通路 在G0/G1期细胞中调节DNA末端切除的程度目前尚不清楚。因此，一个主要的目标是 这个项目是为了发现在G0/G1期调节DNA末端切除的机制和机制 细胞。基于我们的专业知识、新颖的基因筛查方法和 令人信服的初步数据。 基因组维持的另一个关键而又鲜为人知的方面是基因如何表达 在DNA损伤附近的“关闭”，以防止转录和DNA修复之间的碰撞 机械设备。同样，在DNA双链断裂修复后重新启动转录也是至关重要的，但 机制尚不清楚。我们最近用我们的新方法发现了一些与之有关的蛋白质。 和基因筛查，所以这个项目的第二个主要目标是发现 在DNA双链断裂前后，转录关闭和重新启动。

Summary Abstract The overall vision for our research is to discover novel mechanisms by which histone and non-histone proteins on DNA, i.e. chromatin, regulate genomic processes and aging. In particular, we strive to integrate different fields, such as the role of chromatin in genome stability and the role of chromatin in aging. Using a combination of biochemistry, structural biology, molecular genetics in budding yeast, tissue culture and genome-wide approaches, we have discovered that chromatin is disassembled and reassembled during not only gene expression and DNA replication but also during DNA double-strand break repair. We have revealed the mechanistic bases for these events and their key impact on these genomic processes. In more recent years, we have expanded the questions that we address beyond chromatin – for example uncovering novel mechanistic bases of aging and discovering new ways to extend lifespan. Similarly, inspired by our recent finding that chromatin structure reduces the processing of DNA double-strand breaks to single-strand DNA (termed DNA end resection), we have devised innovative CRISPR/Cas9 gRNA library screening approaches to identify novel activities that regulate DNA end resection during DNA double-strand repair. Most of the cells in the human body are in G0/G1-phase and it is critical that excessive DNA end resection does not occur in these cells. If it were to occur, it would block DNA repair by the only pathway that is used to repair DNA double-strand breaks in G0/G1-phase cells, namely non-homologous end joining (NHEJ), and it would result in translocations and deletions from the ensuing homology-mediated repair. Indeed, the extent of DNA end resection is the critical decision point in the choice between using the NHEJ or homologous recombination (HR) pathway for repairing DNA double-strand breaks. We propose that mechanisms must be in place that limit excessive DNA end resection in G0/G1-phase cells to prevent HR, yet enable sufficient DNA end processing of un-ligatable DNA ends to allow NHEJ-mediated repair. The proteins and pathways that regulate the extent of DNA end resection in G0/G1-phase cells are currently unknown. Thus, a major goal of this program is to discover the machinery and mechanisms that regulate DNA end resection in G0/G1-phase cells. We are uniquely positioned to do this, based on our expertise, novel genetic screening approach and compelling preliminary data. Another critical, yet poorly understood, aspect of genome maintenance is how gene expression is “shut-off” in the vicinity of a DNA lesion to prevent collisions between the transcription and DNA repair machinery. Similarly, it is crucial that transcription is restarting after DNA double-strand break repair, but the mechanism is unknown. We have recently discovered some of the proteins involved using our novel assays and genetic screens, so the second major goal of this program is to discover the fundamental mechanisms of transcriptional shut-off and restart around DNA double-strand breaks.