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

调节哺乳动物细胞中 DNA 双链断裂修复事件的新途径

• 批准号: 10093685
• 负责人: Jessica K Tyler
• 金额: $ 42.38万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10093685
• 关键词: Address; Aging; Biochemistry; Biological Assay; CRISPR/Cas technology; Cells; Chromatin; Chromatin Structure; DNA; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA lesion; Data; Double Strand Break Repair; Event; Excision; G1 Phase; Gene Expression; Genetic Diseases; Genetic Screening; Genetic Transcription; Genome; Genome Stability; Genomics; Goals; Guide RNA; Histones; Human Genetics; Human body; Libraries; Longevity; Maintenance; Mammalian Cell; Mediating; Molecular Genetics; Nonhomologous DNA End Joining; Pathway interactions; Positioning Attribute; Process; Proteins; Research; Role; Saccharomycetales; Single-Stranded DNA; Vision; base; ds-DNA; genome integrity; genome-wide; homologous recombination; innovation; non-histone protein; novel; prevent; programs; repaired; screening; structural biology; tissue culture

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.

摘要摘要 我们研究的总体愿景是发现组蛋白和非历史的新型机制 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，例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，萌芽酵母中的分子遗传学，组织培养和 全基因组的方法，我们发现染色质是拆卸和重新组装的 仅基因表达和DNA复制，而且在DNA双链断裂修复过程中。我们有 揭示了这些事件的机械基础及其对这些基因组过程的关键影响。更多 近年来，我们扩大了我们在染色质之外提出的问题 - 例如发现 衰老的新机械基础，并发现延长寿命的新方法。同样，受我们的启发 最近的发现，染色质结构减少了DNA双链破裂的加工到单链 DNA（称为DNA终端切除），我们设计了创新的CRISPR/CAS9 GRNA库筛选 鉴定在DNA双链修复过程中调节DNA终端切除的新型活动的方法。 人体中的大多数细胞都在G0/G1期间，至关重要的DNA末端切除至关重要 这些细胞不存在。如果发生，它将通过唯一用于的途径阻止DNA修复 修复G0/G1相细胞中的DNA双链断裂，即非理论末端连接（NHEJ），IT 将导致确保同源介导的维修的易位和缺失。确实，程度 DNA终端切除术是使用NHEJ或同源之间选择的关键决策点 修复DNA双链断裂的重组（HR）途径。我们建议机制必须是 限制G0/G1相细胞中多余的DNA末端切除以防止HR，但可以启用足够的DNA 最终处理不可粘的DNA末端，以允许NHEJ介导的修复。蛋白质和途径 目前未知，调节G0/G1相细胞中DNA末端切除的程度。那是一个主要目标 该程序是要发现调节G0/G1相调节DNA末端切除的机制和机制 细胞。根据我们的专业知识，新颖的遗传筛查方法，我们有独特的位置 引人入胜的初步数据。 基因组维持的另一个关键但知之甚少的方面是基因表达方式 DNA病变附近的“关闭”，以防止转录和DNA修复之间的碰撞 机械。同样，至关重要的是，在DNA双链破裂修复后，转录正在重新启动，但是 机制是未知的。我们最近发现了使用我们的新测定的一些蛋白质 和遗传筛选，因此该计划的第二个主要目标是发现 转录关闭并重新启动DNA双链断裂。