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: 9361771
• 负责人: MATTHEW J O'CONNELL
• 金额: $ 33.9万
• 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 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9361771
• 关键词: Address; Aging; Bypass; Cancer Biology; Cell Cycle; Cell Cycle Checkpoint; Cell Proliferation; Centromere; Characteristics; Chromatin; Colon; Colon Carcinoma; Complement; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA lesion; DNA replication fork; Data; Degenerative Disorder; Development; Dissociation; Drosophila genus; Ensure; Environment; Enzymes; Event; Excision; Excision Repair; Family; Family member; Fission Yeast; Flap Endonucleases; Frequencies; Genes; Genetic Recombination; Genetic Screening; Genome; Genomics; Genotoxic Stress; Goals; Heterochromatin; Homologous Gene; Human; Immune; Individual; Induced Mutation; Invaded; Lead; Learning; Lesion; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Minor Planets; Modeling; Modification; Mutate; Mutation; Names; Nature; Nonhomologous DNA End Joining; Okazaki fragments; Paper; Pathologic; Pathway interactions; Phosphotransferases; Positioning Attribute; Process; Property; Proteins; Radiation; Recruitment Activity; Regulation; Research; Role; S Phase; Saccharomyces cerevisiae; Signal Transduction; Sister Chromatid; Site; Source; Specificity; Stress; Syndrome; System; Testing; Time; Type I DNA Topoisomerases; Work; Yeasts; cancer therapy; ds-DNA; experimental study; feeding; genetic analysis; genome integrity; homologous recombination; in vivo; loss of function; neoplastic cell; novel; nuclease; nuclease I; prevent; public health relevance; recombinational repair; repaired; response; scaffold; spleen exonuclease; tool

Many forms of DNA damage originate from intrinsic and extrinsic sources. If unrepaired or repaired in an error- prone manner, these lesions induce the mutations and genome rearrangements characteristic of cancer, aging and degenerative diseases. Two particularly pathological lesions are DNA double stranded DNA breaks (DSBs) and collapsed replication forks. The latter originate from stalled replication associated with dissociation of the replisome from the template, and are particularly problematic on the leading strand. To promote the activation of cell cycle checkpoints, the pausing mechanisms to allow time for repair of these lesions and/or replication restart, ssDNA is generated at these sites in a 5'!3' 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 (the name Asteroid derives from the proximity of the gene to the Star locus in Drosophila). These include Ast1 in S. pombe and ASTE1 in humans (there is no Asteroid homolog in Saccharomyces cerevisiae), and ASTE1 is a gene frequently mutated in colon and liver cancers characterized by immune infiltrates, though whether it is a driver or a passenger is not known. We have shown that these nucleases are differentially recruited to DSBs depending on their genomic position, and also depending on the complement of nucleases. Additional studies indicate that the XRNs also function at collapsed replication forks, cooperating with several other enzymes that modulate fork stability and processing. Experiments in this proposal further investigate these phenomena utilizing an armory of new tools to study the processing of these lesions across the genome. We further carry out a thorough analysis of Ast1 to bring the understanding of this conserved enzyme to level commensurate with its long-studied cousins. 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.

许多形式的 DNA 损伤源自内在和外在来源。如果未修复或修复错误- 这些病变很容易诱发癌症、衰老等特征的突变和基因组重排 和退行性疾病。两种特别病理性的损伤是 DNA 双链 DNA 断裂 （DSB）和折叠的复制叉。后者源于与解离相关的复制停滞 复制体的模板，并且在前导链上尤其有问题。为促进 细胞周期检查点的激活、暂停机制以留出时间修复这些损伤和/或 复制重新启动后，ssDNA 在这些位点以 5'！3' 方向生成。由此产生的 ssDNA 仍然存在 具有暴露的 3'-OH 基团，并充当检查点信号复合物组装的着陆垫 以及促进侵入姐妹染色单体的重组酶。 使用裂殖酵母裂殖酵母作为基因和途径发现工具，我们 确定了 XPG 相关核酸酶 (XRN) 家族作为长期追捧的酶，它们是必需的和 足以进行 DSB 端部切除。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中发挥作用。新确定的第三个 这个家族的成员被称为小行星核酸酶（小行星这个名字源自于邻近的核酸酶） 果蝇星基因座的基因）。其中包括粟酒裂殖酵母中的 Ast1 和人类中的 ASTE1（没有 酿酒酵母中的小行星同源物），而 ASTE1 是结肠和肝脏中经常突变的基因 以免疫浸润为特征的癌症，但尚不清楚是司机还是乘客。 我们已经证明，这些核酸酶根据其基因组差异而被招募到 DSB。 位置，并且还取决于核酸酶的补体。其他研究表明 XRN 还 在塌陷的复制叉上发挥作用，与其他几种调节复制叉稳定性的酶合作， 加工。本提案中的实验利用一系列新工具进一步研究这些现象 研究这些病变在基因组中的处理过程。我们进一步对Ast1进行彻底分析 将对这种保守酶的理解提高到与其长期研究的近亲相称的水平。作为 DNA 损伤反应中的这些起始事件会进入许多下游反应途径，这项工作 对确保基因组完整性的许多机制的研究具有重大影响。