Preferential single-strand break repair in the active genes of mammalian cells

哺乳动物细胞活性基因的优先单链断裂修复

• 批准号: 8663322
• 负责人: TAPAS K HAZRA
• 金额: $ 33.13万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8663322
• 关键词: APTX gene; Age; Ataxia; Base Excision Repairs; Brain; Breathing; CAG repeat; Cell Death; Cell Line; Cell Nucleus; Cells; Code; Complex; Coupled; DNA; DNA Damage; DNA Excision Repair Protein ERCC-6; DNA Repair; DNA Single Strand Break; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Defect; Dependency; Development; Disease; Ectopic Expression; Employee Strikes; Enzymes; Foundations; Functional RNA; Functional disorder; Future; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; Health; Heterogeneous-Nuclear Ribonucleoprotein U; Human; Impairment; In Vitro; Inherited; Intervention; Ionizing radiation; Lead; Ligase; Link; MJD1 protein; Machado-Joseph Disease; Mammalian Cell; Mediating; Mitochondria; Mitochondrial RNA; Molecular; Molecular Biology; Molecular Profiling; Multiprotein Complexes; Mus; Mutagens; Mutate; Nerve Degeneration; Nervous system structure; Neuronal Differentiation; Neurons; Nuclear; Nuclear Extract; Nuclear Inclusion; Onset of illness; Organelles; Outcome; Oxidative Stress; Oxygen; Pathogenesis; Pathology; Pathway interactions; Patients; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Polynucleotide 5' -Hydroxyl-Kinase; Population; Prevention strategy; Process; Proteins; RNA Polymerase II; Recombinant Proteins; Role; Single Strand Break Repair; Testing; Time; Tissues; Toxic effect; Transcription-Coupled Repair; Transgenic Mice; base; chemotherapeutic agent; design; gene environment interaction; helicase; in vitro activity; innovation; mitochondrial genome; mutant; nervous system disorder; neuroblastoma cell; neurological pathology; neuronal survival; novel therapeutic intervention; overexpression; plasmid DNA; polyglutamine; prevent; promoter; reconstitution; repair enzyme; repaired; research study; sex; transcription factor

DESCRIPTION (provided by applicant): Gene/environment interactions in the development of various neurological diseases have been well documented. DNA damage, including single-strand breaks (SSBs), is the outcome of one such interaction. Defects in DNA SSB repair (SSBR) may have striking human health consequences. Several neurological diseases have already been identified and characterized that are due to the lack of DNA end-processing activities, catalyzed by enzymes such as aprataxin and TDP1. Human polynucleotide kinase 3'-phosphatase (PNKP) is another SSBR enzyme that processes 3'-P and 5'-OH ends, generated both endogenously and by exogenous genotoxic agents. These DNA termini need to be processed to restore genomic integrity, because unrepaired SSBs would block transcription, which is detrimental in all cells. We hypothesize that SSBs in the transcribed strand of active genes are preferentially repaired via a subpathway of SSBR, which we call transcription-coupled SSBR (TC-SSBR), and that PNKP plays a vital role for 3'-P and 5'-OH end- processing in the transcribed sequences. We have now found that PNKP is present in the mitochondria. The association of PNKP with nuclear and mt RNA polymerases, and preferential association of PNKP with transcribed genes, further supports our hypothesis of preferential repair of actively transcribed genes. Our surprising observation of the association of PNKP with Ataxin-3 (ATXN3), a protein responsible for spinocerebellar ataxia type 3, also called Machado-Joseph Disease (MJD/SCA3), prompted us to investigate PNKP's role in the pathogenesis of the disease. MJD/SCA3 is a fatal, autosomal dominant disorder caused by CAG repeat (poly-Q) expansion in the coding region of the ATXN3 gene. There is no therapy available for this disease. A common pathological feature of poly-Q diseases is the accumulation of intranuclear inclusions. However, the mechanism by which pathogenic ATXN3 (ATXN3-Q72) causes neurodegeneration is still not clearly understood. Our preliminary data showed that the pathological form of ATXN3 blocked PNKP-mediated SSBR activities in vitro. It is thus likely that the pathological form will block PNKP-mediated TC-SSBR as well. The nervous system encounters a high level of oxidative stress, consuming ~20% of inhaled oxygen. Additionally, postmitotic neurons have a high transcriptional rate, which might further increase the dependency of these cells on TC-SSBR to maintain the integrity of both the nuclear and mt genomes. Therefore, to understand the molecular biology of SSBR and the disease process, our project will have three Specific Aims, to test the hypotheses that: 1. ATXN3-Q72 blocks PNKP-mediated nuclear TC-SSBR; 2. ATXN3-Q72 blocks PNKP-mediated mtTC-SSBR; and 3. Ectopic expression of PNKP will rescue ATXN3-Q72-mediated cellular toxicity. Our long-term goal is to determine the mechanistic basis for the development of Ataxia and to develop new strategies for the prevention or treatment of MJD/SCA3 in the human population.

描述（由申请人提供）：基因/环境相互作用在各种神经系统疾病的发展中已经得到了很好的记录。DNA损伤，包括单链断裂（SSBs），就是这样一种相互作用的结果。DNA SSB修复缺陷（SSBR）可能对人类健康造成严重后果。一些神经系统疾病已经被确定和表征，这些疾病是由于缺乏DNA末端加工活性，由aprataxin和TDP1等酶催化。人多核苷酸激酶3′-磷酸酶（PNKP）是另一种处理3′-P和5′-OH末端的SSBR酶，可由内源性和外源性基因毒性物质产生。这些DNA末端需要处理以恢复基因组的完整性，因为未修复的SSBs会阻断转录，这对所有细胞都是有害的。我们假设活性基因转录链中的SSBs通过SSBR的亚通路优先修复，我们称之为转录偶联SSBR (TC-SSBR)，并且PNKP在转录序列中的3'- p和5'- oh末端加工中起着至关重要的作用。我们现在已经发现PNKP存在于线粒体中。PNKP与核和mt RNA聚合酶的关联，以及PNKP与转录基因的优先关联，进一步支持了我们关于活性转录基因优先修复的假设。我们对PNKP与Ataxin-3 (ATXN3)（一种负责脊髓小脑性共济失调3型，也称为Machado-Joseph病（MJD/SCA3）的蛋白质）的关联的惊人观察促使我们研究PNKP在疾病发病机制中的作用。MJD/SCA3是一种致命的常染色体显性遗传病，由ATXN3基因编码区CAG重复（多q）扩增引起。目前尚无治疗这种疾病的方法。多q疾病的一个共同病理特征是核内包涵体的积累。然而，致病性ATXN3 （ATXN3- q72）引起神经退行性变的机制尚不清楚。我们的初步数据显示，病理形式的ATXN3在体外阻断了pnkp介导的SSBR活性。因此，病理形式可能也会阻断pnkp介导的TC-SSBR。神经系统遇到高水平的氧化应激，消耗约20%的吸入氧气。此外，有丝分裂后神经元具有高转录率，这可能进一步增加了这些细胞对TC-SSBR的依赖性，以维持核基因组和mt基因组的完整性。因此，为了了解SSBR的分子生物学和疾病过程，我们的项目将有三个具体目标，以测试假设：1。ATXN3-Q72阻断pnkp介导的核TC-SSBR；2. ATXN3-Q72阻断pnkp介导的mtTC-SSBR；和3。PNKP的异位表达可以缓解atxn3 - q72介导的细胞毒性。我们的长期目标是确定共济失调发展的机制基础，并制定预防或治疗MJD/SCA3人群的新策略。