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DNA Topoisomerase Misfunctioning in Neurological Disease

神经系统疾病中的 DNA 拓扑异构酶功能失调

基本信息

批准号：
10321290
负责人：
Karin Nitiss
金额：
$ 8万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10321290
关键词：
ASD patient Affect Alleles Antineoplastic Agents Biochemical Biological Assay Biological Models Biological Process Cell Differentiation process Cell Line Cell Survival Cells Characteristics Chromosome Segregation Chromosome Structures Complement Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Gene DNA Structure DNA Topoisomerases DNA strand break Data Defect Development Disease Drug Targeting Enzymes Generations Genes Genetic Transcription Goals Human Impairment In Vitro Lead Mammalian Cell Measures Mediating Molecular Conformation Mus Mutation Neuroblastoma Neuronal Differentiation Neurons Patients Pediatric Hospitals Play Process Property Protein Isoforms Proteins Reaction Reporting Role Saint Jude Children&apos s Research Hospital Testing Topoisomerase Topoisomerase II Transcription Initiation Work Yeasts autism spectrum disorder brain dysfunction cell growth cell type embryonic stem cell expression vector gene repair human disease knock-down loss of function loss of function mutation mouse model mutant nervous system disorder neuron development neuron loss neuronal survival neurotoxic phosphodiester preservation repaired response small hairpin RNA small molecule inhibitor

项目摘要

DNA topoisomerase II (Top2) carries out changes in DNA structure needed for efficient transcription, replication, and DNA repair. This enzymes introduce transient double strand breaks in DNA through a protein/DNA covalent intermediate termed the cleavage complex. The DNA cleavage mechanism of Top2 allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks. Mammalian cells contain two Top2 isoforms termed Top2α and Top2β. The two enzymes have distinct biological functions with Top2β having unique roles in transcription and chromosome structure. Top2β is particularly important for transcription of neuronal genes. Interestingly, topoisomerases have recently been suggested to be particularly important in the transcription of long genes, suggesting that topoisomerase function may be uniquely important in neuronal cells. While the Top2 catalytic mechanism typically avoids generating double strand breaks, in some contexts, it has been suggested that Top2β makes long lasting double strand breaks during the process of regulated transcription initiation. Recent results have also suggested that long lasting topoisomerase covalent complexes may cause neurotoxic DNA damage. Two recent reports have identified identical heterozygous Top2β mutations in patients with autism spectrum disorders (ASDs). In preliminary data, we show that the Top2β mutation found in the two independent ASD patients generates spontaneous DNA damage through the generation of enzyme mediated DNA strand breaks. In this application, we propose to explore the biochemical characteristics of this type of enzyme using in vitro enzyme assays and expression of the mutant enzyme in yeast and mammalian cells. A second aim of our studies is to study DNA damaging Top2β proteins in neuronal cells. Our collaborator Peter McKinnon, St. Jude Children's Hospital, has generated mouse ES cells that express two distinct Top2β mutations that in vitro lead to elevated topoisomerase mediated cleavage. We plan to characterize DNA damage responses and developmental defects when these cells differentiate into neuronal lineages. Finally, we propose to initiate development of a model system using human neuroblastoma SH-SY5Y cells to introduce topoisomerase mutations capable of generating spontaneous DNA damage into human neuronal cells. We will determine whether DNA damaging mutations of Top2β have similar effects to loss of function mutations, or whether the generation of topoisomerase induced DNA damage leads to unique effects on neuronal cell differentiation and survival. These studies will highlight potential roles of endogenous DNA damage in human neurological diseases, and provide a model system that can be used to explore unique ways that topoisomerase mis- functioning can contribute to human diseases.

DNA拓扑异构酶II （Top2）进行有效转录所需的DNA结构改变，