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损伤起源于内在和外在来源。如果未修理或修理错误- 倾向的方式，这些病变诱导突变和基因组重排特征的癌症，衰老， 和退行性疾病。两种特别的病理损伤是DNA双链DNA断裂 （DSB）和折叠的复制分叉。后者起源于与解离相关的停滞复制 复制体从模板中分离出来，特别是在前导链上。推进 细胞周期检查点的激活，允许这些损伤修复时间的暂停机制，和/或 复制重新启动，ssDNA在这些位点以5 '！3'方向。剩下的ssDNA 具有暴露的3 '-OH基团，并作为组装检查点信号传导复合物的着陆垫， 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， 确定了一个XPG相关核酸酶家族（XRN），作为长期寻求的必需酶， 足以在DSB处进行末端切除。这包括已知已久的Rad 2/Fen 1和Exo 1酶， 在冈崎片段成熟和各种切除修复途径中发挥作用。新发现的第三 这个家族的成员被称为小行星核酸酶（名称小行星来自于接近的小行星）。 果蝇中的星星基因座）。其中包括S. pombe和ASTE 1在人类（没有 ASTE 1是结肠和肝脏中经常发生突变的基因 以免疫浸润为特征的癌症，尽管不知道是司机还是乘客。 我们已经表明，这些核酸酶根据其基因组差异被差异地募集到DSB。 位置，也取决于核酸酶的互补性。进一步的研究表明，XRN还 在折叠的复制叉上起作用，与调节叉稳定性的几种其他酶合作， 处理.实验在这个建议进一步调查这些现象利用军械库的新工具 来研究这些病变在基因组中的处理过程。我们进一步对Ast 1进行了深入的分析， 将对这种保守酶的理解提升到与其长期研究的表亲相当的水平。作为 这些DNA损伤反应的起始事件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许多机制具有重大影响。