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.

多种形式的DNA损伤起源于固有和外在来源。如果未修复或在错误中修复或修复 这些病变俯卧，诱导癌症的突变和基因组重排特征，衰老 和退化性疾病。两个特别的病理病变是DNA双链DNA断裂 （DSB）和折叠复制叉。后者起源于与解离相关的停滞复制 来自模板的重新组合，在领先链上尤其有问题。促进 细胞周期检查点的激活，暂停机制，使这些病变的时间和/或 复制重新启动，ssDNA是在5'！3'方向上生成的。剩下的ssDNA 有一个暴露的3'-OH组，充当装配检查点信令复合物的降落垫作为 以及促进侵入姐妹染色单体的重组酶。 使用裂变酵母菌杂种菌Pombe作为基因和途径发现工具，我们 识别出XPG相关核酸酶（XRN）家族是长期以来一直在寻求必要的酶，并且 足以在DSB的最终切除术。这是由已知已知的RAD2/FEN1和EXO1酶组成的 在Okazaki碎片成熟和各种切除修复途径中的功能。新确定的第三 该家族的成员被称为小行星核酸酶（小行星的名称源自 基因到果蝇中的恒星基因座。其中包括S. pombe和Aste1中的AST1（没有 酿酒酵母中的小行星同源物）和ASTE1是在结肠和肝脏中经常突变的基因 以免疫浸润为特征的癌症，尽管它是驾驶员还是乘客。 我们已经表明，这些核酸酶根据其基因组而差异化为DSB 位置，也取决于核酸酶的补充。其他研究表明XRN也 折叠复制叉的功能，与调节叉稳定性和的其他几种酶合作 加工。该提案中的实验进一步研究了这些现象，利用新工具的军械库 研究这些病变在整个基因组中的处理。我们进一步对AST1进行了彻底的分析 使对这种保守酶的理解与长期研究的表亲相称。作为 这些在DNA损伤反应中的启动事件源于许多下游响应途径，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。