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Processing of lesions into DNA repair and checkpoint pathways

将病变处理为 DNA 修复和检查点通路

基本信息

批准号：
10595083
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
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10595083
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 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 Resected Role S phase Saccharomyces cerevisiae Saccharomycetales Signal Transduction Single-Stranded DNA Sister Chromatid Site Source Specificity Surgical incisions Syndrome System Testing Time Type I DNA Topoisomerases Work Yeasts biological adaptation to stress cancer therapy 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

项目摘要

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，既有内在的，也有外在的。这些损伤可能会导致导致癌症、衰老和退化性疾病的突变和基因组重排。尤其是病理损伤是DNA双链DNA断裂(DSB)以及塌陷的复制叉状结构由复制障碍造成的，这本身就可以促进DSB的形成。为了激活细胞周期检查点，在允许时间修复这些损伤的机制中，单链DNA在这些位置以5‘à3’的方向产生。得到的单链DNA仍然有一个暴露的3‘-OH基团，并作为组装的着陆垫检查点信号复合体以及促进姐妹细胞侵袭的重组酶染色单体。利用裂解酵母菌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，是人们长期追求的必要和充分的酶用于DSB的末端切除。它由已知的Rad2/Fen1和Exo1酶组成，它们也在 Okazaki片段的成熟和各种切除修复途径。这个新确定的第三个成员该家族被称为小行星核酸酶。这些基因包括S.pombe中的Ast1和人类中的ASTE1，但在发芽酵母中没有发现小行星同源物。因此，Ast1酶仍然很差。与Fen1和Exo1相比具有特征性。导致和在这笔赠款的初始资助期内的研究表明，这些XRN 核酸酶被层次化地招募到DSB中，这是动态的，取决于核酸酶的补体。在受损的基因座上，转录方向还有进一步的特异性。相当数量的数据还表明，XRN与其他几种酶协同作用，在折叠的复制分叉中起关键作用这调节了叉子的稳定性和加工。该方案中的其他实验建立在这些观察结果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。我们向您介绍一种彻底分析Ast1，使人们对这种保守的酶的了解达到与其相称的水平长期研究近亲，然后确定他们之间的特异性决定因素。因为DNA中的这些启动事件损伤反应被馈入许多下游反应途径，这一工作对研究确保基因组完整性的许多机制。