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: 8373393
• 负责人: TAPAS K HAZRA
• 金额: $ 33.47万
• 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/8373393
• 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 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. PUBLIC HEALTH RELEVANCE: Machado-Joseph disease, or spinocerebellar ataxia type 3 (MJD/SCA3), is the most common dominantly inherited ataxia worldwide; however, no therapy is available because the molecular mechanism responsible for the disease is not clearly understood. Our study is aimed at identifying a new subpathway for preferential repair of single-strand DNA breaks in the transcribed genes, and showing that deficiencies in this pathway could play a causal role in SCA3/MJD. Our expected results should ultimately lead to new therapeutic intervention strategies, or even to approaches for preventing the onset of diseases etiologically linked to DNA damage and repair.

描述（由申请人提供）：各种神经系统疾病发展中的基因/环境相互作用已得到充分记录。 DNA 损伤，包括单链断裂 (SSB)，就是此类相互作用的结果。 DNA SSB 修复 (SSBR) 缺陷可能会对人类健康产生惊人的影响。一些神经系统疾病已经被鉴定和表征，这些疾病是由于缺乏由 aprataxin 和 TDP1 等酶催化的 DNA 末端加工活性造成的。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，由内源性和外源性基因毒性剂产生。这些 DNA 末端需要进行处理以恢复基因组完整性，因为未修复的 SSB 会阻碍转录，这对所有细胞都是有害的。我们假设活性基因转录链中的 SSB 优先通过 SSBR 的子通路进行修复，我们将其称为转录偶联 SSBR (TC-SSBR)，并且 PNKP 在转录序列中的 3'-P 和 5'-OH 末端加工中发挥着至关重要的作用。我们现在发现 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，进一步支持了我们优先修复活跃转录基因的假设。我们令人惊讶地观察到 PNKP 与 Ataxin-3 (ATXN3) 的关联，Ataxin-3 (ATXN3) 是一种导致 3 型脊髓小脑共济失调的蛋白质，也称为马查多-约瑟夫病 (MJD/SCA3)，促使我们研究 PNKP 在该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疾病，由 ATXN3 基因编码区的 CAG 重复 (poly-Q) 扩展引起。对于这种疾病没有可用的治疗方法。 Poly-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仍不清楚。我们的初步数据表明，ATXN3 的病理形式在体外阻断了 PNKP 介导的 SSBR 活性。因此，病理形式也可能会阻断 PNKP 介导的 TC-SSBR。神经系统面临高水平的氧化应激，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，这可能进一步增加这些细胞对 TC-SSBR 的依赖性，以维持核和线粒体基因组的完整性。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，以检验以下假设： 1. ATXN3-Q72阻断PNKP介导的核TC-SSBR； 2. ATXN3-Q72 阻断 PNKP 介导的 mtTC-SSBR； 3. PNKP 的异位表达将挽救 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，并制定预防或治疗人群 MJD/SCA3 的新策略。 公共卫生相关性：马查多-约瑟夫病或脊髓小脑性共济失调 3 型 (MJD/SCA3) 是全世界最常见的显性遗传性共济失调；然而，由于尚不清楚导致该疾病的分子机制，因此没有可用的治疗方法。我们的研究旨在确定一种新的亚途径，用于优先修复转录基因中的单链 DNA 断裂，并表明该途径的缺陷可能在 SCA3/MJD 中发挥因果作用。我们的预期结果最终应该会导致新的治疗干预策略，甚至是预防与 DNA 损伤和修复有关的疾病发生的方法。