DNA Double Strand Break Repair Deficiency and Neurodegeneration

DNA 双链断裂修复缺陷和神经退行性变

• 批准号: 10161868
• 负责人: TAPAS K HAZRA
• 金额: $ 39.26万
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10161868
• 关键词: Adult; Affect; Apoptosis; Ataxia; Blood - brain barrier anatomy; Brain; Brain region; C-terminal; CAG repeat; Cells; Code; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA strand break; DNA-dependent protein kinase; Data; Defect; Development; Disease; Disease Progression; Double Strand Break Repair; Enzymes; Fructose; G2 Phase; Gel; Genes; Genome; Genomic Instability; Genomic Segment; Glutamine; Glycolysis; Goals; Human; Hybrids; In Vitro; Individual; Inherited; Knowledge; Lesion; Link; MJD1 protein; Machado-Joseph Disease; Mammalian Cell; Measures; Mediating; Modality; Molecular; Multiprotein Complexes; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nonhomologous DNA End Joining; Nuclear Extract; Pathogenesis; Pathway interactions; Patients; Phase; Phosphoric Monoester Hydrolases; Physiological; Play; Polymerase; Polynucleotide 5' -Hydroxyl-Kinase; Process; Protein Splicing; Proteins; RNA; RNA Binding; RNA Polymerase II; RNA-Binding Proteins; RNA-Directed DNA Polymerase; Repair Complex; Reporting; Role; Site; Testing; Therapeutic; Therapeutic Intervention; Tissues; Work; XRCC1 gene; XRCC4 gene; base; brain tissue; design; genetic regulatory protein; homologous recombination; human disease; induced pluripotent stem cell; insight; mutant; nerve stem cell; nervous system disorder; neurotoxicity; novel therapeutic intervention; overexpression; p53-binding protein 1; plasmid DNA; polyglutamine; prevent; promoter; repaired; response; restoration; targeted delivery; therapeutically effective; tool

Spinocerebellar ataxia type 3 (SCA3), aka Machado-Joseph Disease is the most common dominantly inherited ataxia worldwide. It is the result of a CAG (glutamine) repeat expansion in the coding region of Ataxin 3, a polyglutamine (14-41 repeats)-containing protein. While investigating the mechanism of SCA3, we have found that wild type Ataxin 3 stimulates, while the mutant form abrogates the activity of polynucleotide kinase 3’- phosphatase (PNKP), an essential DNA repair protein. This resulted in the accumulation of DNA double-strand breaks in the brains of SCA3 patients and mice. Constant activation of the DNA damage-response pathway with consequent cellular apoptosis is a plausible cause of SCA3. Our recent studies have revealed that PNKP plays a critical role in DNA double strand break repair via classical non-homologous end joining (C-NHEJ). We have demonstrated that PNKP-mediated C-NHEJ repair pathway is error-free, with homologous nascent RNA providing the template to restore the missing sequence at the double strand break site in transcribed genes. DNA strand break analysis in the SCA3 mouse brain versus wild type showed significantly more strand breaks in the transcribed but, not the non-transcribed genes. Collectively, these data indicate that differential genomic region-specific strand break repair occurs in neuronal cells. Our lab found that, in addition to ATXN3, two RNA- binding/splicing proteins (NONO and SFPQ) and a key allosteric regulator of glycolysis are involved in this pathway. We are the first to show the roles of Ataxin 3 and the glycolysis-regulatory protein in classical non- homologous end joining mediated DNA double strand break repair. Notably, all these factors form a physiological complex with RNA polymerase II and other classical non-homologous end joining proteins. We also found that NONO and SFPQ significantly stimulated PNKP’s end-processing activity. We postulate that these RNA-binding proteins facilitate the formation of the RNA-DNA hybrid so that the RNA-dependent DNA polymerase can effectively use the RNA as a template to restore the missing information. However, the glycolysis inducer did not affect PNKP’s activity, but the inducer-catalyzed metabolite significantly stimulated PNKP’s activity. Furthermore, the metabolite can even restore the activity of PNKP in SCA3 patients’ brain nuclear extract, suggesting the promising therapeutic potential of this natural metabolite for SCA3. Hence, this project will test the hypotheses that: restoration of the PNKP-mediated C-NHEJ repair pathway is crucial for maintaining the integrity of the transcribed genome, and thereby rescuing neuronal cells from the deleterious effects of mutant ATXN3. Understanding the mechanistic basis of error-free double strand break repair of the transcribed genes in neuronal cells via classical non-homologous end joining pathway, and the modulatory effect of a natural metabolite in such a pathway, will significantly advance our knowledge and should accelerate the development of new treatment modalities for SCA3 and other polyQ diseases.

脊髓小脑性共济失调3型（SCA 3），又名Machado-Joseph病是最常见的显性遗传性疾病， 全球性共济失调它是共济失调蛋白3编码区CAG（谷氨酰胺）重复扩增的结果， 多聚谷氨酰胺（14-41个重复）蛋白。在研究SCA 3的机制时，我们发现 野生型共济失调蛋白3刺激，而突变形式消除多核苷酸激酶3 '- 磷酸酶（PNKP），一种重要的DNA修复蛋白。这导致了DNA双链的积累 SCA 3患者和小鼠大脑中的断裂。DNA损伤反应途径的持续激活， 随后的细胞凋亡是SCA 3的合理原因。我们最近的研究表明PNKP 通过经典的非同源末端连接（C-NHEJ）在DNA双链断裂修复中起关键作用。我们有 证明PNKP介导的C-NHEJ修复途径是无错误的，具有同源的新生RNA， 提供模板以恢复转录基因中双链断裂位点处的缺失序列。 SCA 3小鼠脑中的DNA链断裂分析与野生型相比显示出显著更多的链断裂 在转录的基因中，而不是在非转录的基因中。总的来说，这些数据表明，差异基因组 区域特异性链断裂修复发生在神经元细胞中。我们的实验室发现，除了ATXN 3，两种RNA- 结合/剪接蛋白（NONO和SFPQ）和糖酵解的关键变构调节因子参与其中 通路我们是第一个显示Ataxin 3和糖酵解调节蛋白在经典的非胰岛素依赖型糖尿病中的作用。 同源末端连接介导的DNA双链断裂修复。值得注意的是，所有这些因素都形成了一种生理上的 与RNA聚合酶II和其他经典的非同源末端连接蛋白复合。我们还发现 NONO和SFPQ显着刺激PNKP的最终加工活动。我们假设这些RNA结合 蛋白质促进RNA-DNA杂合体的形成，使得RNA依赖性DNA聚合酶可以 有效地使用RNA作为模板来恢复丢失的信息。然而，糖酵解诱导剂没有 影响PNKP的活性，但诱导物催化的代谢产物显著刺激PNKP的活性。此外，委员会认为， 代谢产物甚至可以恢复SCA 3患者脑核提取物中PNKP的活性，这表明 这种天然代谢物对SCA 3的有希望的治疗潜力。因此，本项目将测试假设 PNKP介导的C-NHEJ修复途径的恢复对于维持细胞的完整性至关重要。 转录的基因组，从而拯救神经细胞免受突变体的有害影响， ATXN3。理解转录基因无错双链断裂修复的机制基础 在神经元细胞中通过经典的非同源末端连接途径，以及天然的 在这样的途径中的代谢物，将显着推进我们的知识，并应加速发展 SCA 3和其他polyQ疾病的新治疗模式。

项目成果

Highly Sensitive Radioactivity-Based DNA 3'-Phosphatase Activity Assay for Polynucleotide Kinase 3'-Phosphatase.

基于放射性的高灵敏 DNA 3-磷酸酶活性测定，用于检测多核苷酸激酶 3-磷酸酶。

• DOI: 10.1007/978-1-0716-3373-1_3
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Chakraborty,Anirban;Hazra,TapasK
• 通讯作者: Hazra,TapasK

Synergy of melanin and vitamin-D may play a fundamental role in preventing SARS-CoV-2 infections and halt COVID-19 by inactivating furin protease.

• DOI: 10.1186/s41231-020-00073-y
• 发表时间: 2020
• 期刊: Translational medicine communications
• 作者: Paria K;Paul D;Chowdhury T;Pyne S;Chakraborty R;Mandal SM
• 通讯作者: Mandal SM

Innate Immune Responses to RSV Infection Facilitated by OGG1, an Enzyme Repairing Oxidatively Modified DNA Base Lesions.

OGG1（一种修复氧化修饰 DNA 碱基损伤的酶）促进对 RSV 感染的先天免疫反应

• DOI: 10.1159/000524186
• 发表时间: 2022
• 期刊: Journal of innate immunity
• 影响因子: 5.3

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

• DOI: 10.1371/journal.pgen.1004834
• 发表时间: 2015-01
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Gao R;Liu Y;Silva-Fernandes A;Fang X;Paulucci-Holthauzen A;Chatterjee A;Zhang HL;Matsuura T;Choudhary S;Ashizawa T;Koeppen AH;Maciel P;Hazra TK;Sarkar PS
• 通讯作者: Sarkar PS