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: 10557230
• 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/10557230
• 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组蛋白的新机制 DNA 上的蛋白质（即染色质）调节基因组过程和衰老。 不同领域，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。 生物化学、结构生物学、芽殖酵母分子遗传学、组织培养和 通过全基因组方法，我们发现染色质在非 不仅是基因表达和 DNA 复制，而且还包括 DNA 双链断裂修复过程。 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。 近年来，我们将解决的问题扩展到染色质之外——例如揭示 衰老的新颖机制基础和发现延长寿命的新方法同样受到我们的启发。 最近发现染色质结构减少了 DNA 双链断裂到单链的加工 DNA（称为DNA末端切除），我们设计了创新的CRISPR/Cas9 gRNA文库筛选 方法来识别在 DNA 双链修复过程中调节 DNA 末端切除的新活性。 人体大部分细胞都处于G0/G1期，过度的DNA末端切除至关重要 这些细胞中不会发生这种情况，如果发生的话，它会通过唯一的途径来阻止 DNA 修复。 修复G0/G1期细胞中的DNA双链断裂，即非同源末端连接（NHEJ）， 事实上，会导致随后的同源介导的修复的易位和缺失。 DNA 末端切除的效果是选择使用 NHEJ 或同源 DNA 的关键决策点 我们提出修复 DNA 双链断裂的重组 (HR) 途径。 限制 G0/G1 期细胞中过度 DNA 末端切除以防止 HR，但仍能提供足够的 DNA 对不可连接的 DNA 末端进行末端处理，以实现 NHEJ 介导的修复。 调节 G0/G1 期细胞 DNA 末端切除的程度目前尚不清楚。 该计划旨在发现调节 G0/G1 期 DNA 末端切除的机制和机制 凭借我们的专业知识、新颖的基因筛查方法和技术，我们拥有独特的优势来做到这一点。 令人信服的初步数据。 基因组维护的另一个关键但仍知之甚少的方面是基因表达是如何进行的 在 DNA 损伤附近“关闭”，以防止转录和 DNA 修复之间的冲突 同样，DNA 双链断裂修复后转录的重新开始也至关重要，但 我们最近使用我们的新检测方法发现了一些涉及的蛋白质。 和基因筛选，所以这个项目的第二个主要目标是发现的基本机制 DNA 双链断裂周围的转录关闭和重启。