Processing of lesions into DNA repair and checkpoint pathways

将病变处理为 DNA 修复和检查点通路

• 批准号: 10375441
• 负责人: MATTHEW J O'CONNELL
• 金额: $ 34.79万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10375441
• 关键词: Aging; Bypass; Cancer Biology; Catalysis; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Proliferation; Centromere; Chromatin; Complement; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA lesion; DNA replication fork; Data; Degenerative Disorder; Development; Ensure; Environment; Enzymes; Event; Excision; Excision Repair; Family; Family member; Fission Yeast; Frequencies; Funding; Genes; Genetic Recombination; Genetic Screening; Genetic Transcription; Genome; Genomics; Genotoxic Stress; Goals; Grant; Heterochromatin; Homologous Gene; Human; Induced Mutation; Invaded; Lead; Learning; Lesion; Malignant Neoplasms; Mediating; Minor Planets; Modeling; Nonhomologous DNA End Joining; Okazaki fragments; Organism; Paper; Pathologic; Pathway interactions; Phosphotransferases; Process; Property; Proteins; RAD52 gene; Reaction; Regulation; Role; S phase; Saccharomyces cerevisiae; Saccharomycetales; Signal Transduction; Sister Chromatid; Site; Source; Specificity; Surgical incisions; Syndrome; System; Testing; Time; Type I DNA Topoisomerases; Work; Yeasts; biological adaptation to stress; cancer therapy; ds-DNA; endonuclease; experimental study; genetic analysis; genome integrity; homologous recombination; in vivo; loss of function; neoplastic cell; novel; nuclease; nuclease I; preservation; prevent; public health relevance; recruit; repaired; replication stress; response; restraint; scaffold; tool

DNA damage comes in many forms that originate from intrinsic and extrinsic sources. These lesions can induce the mutations and genome rearrangements that lead to cancer, aging and degenerative diseases. Particularly pathological lesions are the DNA double stranded DNA breaks (DSBs), as well as collapsed replication forks caused by barriers to replication, that can themselves promote DSB formation. To activate cell cycle checkpoints, mechanisms that allow the time to repair these lesions, ssDNA is generated at these sites in a 5’à3’ direction. The resulting ssDNA that remains has an exposed 3’-OH group, and acts as a landing pad for assembly of checkpoint signaling complexes as well as recombination enzymes that promote invasion into the sister chromatid. Using the fission yeast Schizosaccharomyces pombe as a gene and pathway discovery tool, we identified a family of XPG-related nucleases (XRNs) as the long sought after enzymes that are necessary and sufficient for end resection at DSBs. This consists of the long known Rad2/Fen1 and Exo1 enzymes that also function in Okazaki Fragment maturation and various Excision Repair pathways. The newly identified third member of this family is known as the Asteroid nucleases. These include Ast1 in S. pombe and ASTE1 in humans, but there is no Asteroid homolog in the budding yeast Saccharomyces cerevisiae. Thus, Ast1 enzymes remain poorly characterized compared to Fen1 and Exo1. Studies leading to, and during the initial funding period of this grant, have shown that these XRN nucleases are hierarchically recruited to DSBs, which is dynamic depending on the complement of nucleases. There is further specificity afforded by the direction of transcription at a damaged locus. A considerable body of data also shows that the XRNs are critical at collapsed replication forks, cooperating with several other enzymes that modulate fork stability and processing. Additional experiments in this proposal build on these observations and utilize an armory of new tools to study the processing of these lesions across the genome. We present a thorough analysis of Ast1 to bring the understanding of this conserved enzyme to level commensurate with its long-studied cousins, then determine specificity determinants among them. As these initiating events in DNA damage responses feed into many downstream response pathways, this work has significant impact on the study of the many mechanisms that ensure the integrity of the genome.

DNA 损伤有多种形式，有内在的也有外在的。这些病变可诱发 导致癌症、衰老和退行性疾病的突变和基因组重排。特别 病理损伤是DNA双链DNA断裂（DSB），以及折叠的复制叉 由复制障碍引起的，其本身可以促进 DSB 的形成。为了激活细胞周期检查点， 机制允许有时间修复这些损伤，单链 DNA 在这些位点以 5'à3' 方向生成。 剩下的 ssDNA 具有暴露的 3'-OH 基团，并充当组装的着陆垫 检查点信号复合物以及促进入侵姐妹的重组酶 染色单体。 使用裂殖酵母裂殖酵母作为基因和途径发现工具，我们发现 XPG 相关核酸酶 (XRN) 家族是长期寻求的必要且充分的酶 用于 DSB 的末端切除。它由众所周知的 Rad2/Fen1 和 Exo1 酶组成，它们也在 冈崎片段成熟和各种切除修复途径。新确定的第三位成员 该家族被称为小行星核酸酶。其中包括粟酒裂殖酵母中的 Ast1 和人类中的 ASTE1，但也有 芽殖酵母酿酒酵母中没有 Asteroid 同源物。因此，Ast1酶仍然很差 与 Fen1 和 Exo1 相比进行了表征。 此项资助的初始资助期间的研究表明，这些 XRN 核酸酶被分层招募到 DSB，其动态取决于核酸酶的补体。 受损位点的转录方向提供了进一步的特异性。相当大的身体 数据还表明，XRN 与其他几种酶合作，对于折叠复制叉至关重要 调节货叉的稳定性和加工能力。本提案中的其他实验建立在这些观察的基础上 并利用一系列新工具来研究这些病变在基因组中的处理过程。我们提出一个 对 Ast1 的彻底分析，使人们对这种保守酶的理解达到与其 长期研究的表亲，然后确定它们之间的特异性决定因素。由于 DNA 中的这些起始事件 损伤反应进入许多下游反应途径，这项工作对 研究确保基因组完整性的多种机制。