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，来源于内在和外在来源。这些损伤会导致 导致癌症、衰老和退行性疾病的突变和基因组重排。特别 病理性损伤是DNA双链DNA断裂（DSB），以及复制叉塌陷 由复制障碍引起，它们本身可以促进DSB的形成。为了激活细胞周期检查点， 由于这些机制允许有时间修复这些损伤，ssDNA在这些位点以5 'à3'方向产生。 剩余的所得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重组酶，促进入侵到姐妹 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，我们鉴定了 XPG相关核酸酶（XRN）家族，作为长期寻求的酶， 在DSB进行末端切除。这包括长期已知的Rad 2/Fen 1和Exo 1酶，它们也在 冈崎片段成熟和各种切除修复途径。新发现的第三名成员 家族被称为小行星核酸酶。其中包括S. pombe和ASTE 1在人类，但有 在芽殖酵母酿酒酵母中没有类似的小行星。因此，Ast 1酶仍然很差， 与Fen 1和Exo 1相比， 研究导致，并在此赠款的初始资助期间，已经表明，这些XRN 核酸酶被分级地募集到DSB，这是动态的，取决于核酸酶的补充。 在受损基因座的转录方向提供了进一步的特异性。的大量 数据还表明，XRN在复制叉崩溃时至关重要，与其他几种酶合作 调节叉子的稳定性和加工过程。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过程。我们提出了一个 Ast 1的彻底分析，使这种保守的酶的理解水平相称， 长期研究的表亲，然后确定它们之间的特异性决定因素。当DNA中的这些起始事件 损伤响应馈送到许多下游响应途径，这项工作对 研究确保基因组完整性的许多机制。