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

摘要 我们研究的总体愿景是发现组蛋白和非组蛋白相互作用的新机制。 DNA上的蛋白质，即染色质，调节基因组过程和衰老。特别是，我们努力整合 不同的领域，如染色质在基因组稳定性中的作用和染色质在衰老中的作用。使用 结合生物化学，结构生物学，分子遗传学在芽殖酵母，组织培养和 通过全基因组的方法，我们发现染色质在非细胞周期中被分解和重新组装， 不仅基因表达和DNA复制，而且在DNA双链断裂修复期间。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。更 近年来，我们已经扩展了我们解决的问题，超越了染色质-例如发现 衰老的新机制基础和发现延长寿命的新方法。同样，受我们 最近发现染色质结构减少了DNA双链断裂到单链的过程 DNA（称为DNA末端切除），我们设计了创新的CRISPR/Cas9 gRNA文库筛选 方法，以确定新的活动，调节DNA双链修复过程中的DNA末端切除。 人体内的大多数细胞处于G 0/G1期，过量的DNA末端切除至关重要， 并不发生在这些细胞中。如果发生这种情况，它将阻止DNA修复的唯一途径， 修复G 0/G1期细胞中的DNA双链断裂，即非同源末端连接（NHEJ）， 将导致随后同源介导修复的易位和缺失。事实上， DNA末端切除是选择使用NHEJ或同源 重组（HR）途径修复DNA双链断裂。我们建议，必须建立机制， 在G 0/G1期细胞中进行有限过量DNA末端切除以防止HR，但仍能使足够的DNA 末端加工不可连接的DNA末端以允许NHEJ介导的修复。蛋白质和途径， 调节G 0/G1期细胞中DNA末端切除的程度目前尚不清楚。因此， 该项目旨在发现调控G 0/G1期DNA末端切除的机制 细胞基于我们的专业知识、新颖的基因筛选方法和 令人信服的初步数据。 基因组维持的另一个关键但知之甚少的方面是基因表达是如何影响 在DNA损伤附近“关闭”，以防止转录和DNA修复之间的碰撞 机械.类似地，在DNA双链断裂修复后重新启动转录是至关重要的，但是 机制不明。我们最近发现了一些蛋白质参与使用我们的新的分析 和基因筛选，所以这个计划的第二个主要目标是发现 转录关闭和DNA双链断裂周围的重新启动。

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.