Deciphering the mechanisms of PARP1 activity in telomere integrity

破译 PARP1 活性在端粒完整性中的机制

• 批准号: 9320953
• 负责人: Elise Fouquerel
• 金额: $ 8.91万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9320953
• 关键词: Address; Adenosine Diphosphate Ribose; Affinity; Aging; Aphidicolin; Binding; Biological Assay; Biological Preservation; Biological Process; Biology; Cells; Chromatin; Chromosomes; Co-Immunoprecipitations; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA biosynthesis; DNA replication fork; Development; Disease; Environmental Exposure; Enzyme Inhibitor Drugs; Enzymes; Exposure to; Foundations; Functional disorder; G-Quartets; Genetic Transcription; Genome; Genome Stability; Genomic DNA; Genotoxic Stress; Goals; Health; Histones; Human; Hydrogen Peroxide; Immunofluorescence Immunologic; Individual; Inflammatory; Lead; Length; Ligands; Maintenance; Malignant Neoplasms; Measures; Methylnitronitrosoguanidine; Monitor; Mutate; Nicotinamide adenine dinucleotide; Nucleoproteins; Oxidative Stress; Pathologic; Play; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymers; Population; Positioning Attribute; Protein Subunits; Proteins; RNA-Directed DNA Polymerase; Recruitment Activity; Regulation; Reporting; Research; Role; Signal Transduction; Sister Chromatid Exchange; Somatic Cell; Stem cells; Stimulus; Stress; Structure; System; TERF1 gene; TINF2 gene; Telomerase; Telomere Length Maintenance; Telomere Maintenance; Telomere Shortening; Telomeric Repeat Binding Protein 2; Testing; Therapeutic; Training; Transducers; Work; cancer cell; cell growth regulation; design; environmental agent; experience; genotoxicity; helicase; homologous recombination; in vitro activity; in vivo; innovation; interest; member; mutant; novel therapeutic intervention; prevent; programs; protein complex; repaired; response; single molecule; telomere; telomere loss

Deciphering the mechanisms of PARP1 activity in telomere integrity The goals of this project are to define poly(ADP-ribose) polymerase 1 (PARP1) roles in telomere preservation under normal conditions and after environmental genotoxic exposure. My strong background in the PARP field and my ongoing training in telomere biology, place me in a unique position to successfully complete this project. In the long term, this project will lay the foundation for me to establish an independent research program investigating roles for PARPs in genome stability and human health. Telomeres consist of TTAGGG repeat arrays bound by the 6-member shelterin protein complex, and are lengthened by the reverse transcriptase telomerase in stem cells and most cancer cells. Telomere attrition and dysfunction can arise from exposure to various environmental agents and are associated with aging-related diseases and cancer. Previous studies have implicated PARP1 in telomere maintenance, but the mechanisms are poorly understood. PARP1 synthesizes poly(ADP-ribose) (PAR) and this activity is critical for genome maintenance by facilitating DNA repair pathways and regulating DNA replication during replication stress. The central hypothesis of this proposal is that PARP1 preserves telomere integrity by modulating the activities of the telomere shelterin proteins and telomerase. In support of this, I demonstrated that shelterin proteins as well as telomerase, bind to PAR. I also observed that PAR binding stimulates telomerase activity in vitro. Aim 1 will define interactions between PARP1 and the various shelterin proteins under normal conditions and after genotoxic insult, and the roles for these interactions in telomeric DNA repair. We will test for PARylation of shelterin proteins, and will define their affinity for PAR. Oxidative and alkylating DNA damage will be induced with H2O2 and MNNG respectively, and 8-oxoguanine will be locally induced at telomeres using an innovative targeting system. We will follow PARP1 recruitment to telomeres after DNA damage, and will measure DNA repair rates and telomere aberrations in PARP1 proficient and deficient cells. Aim 2 will examine PARP1 roles in regulating telomerase. We will examine PAR binding to reported putative PAR binding motifs (PBMs) in the hTERT subunit, and will examine the effect on telomerase activity by mutating these PBMs. We will monitor telomere length alterations in cells expressing wild type and PBM mutant hTERT, and telomerase recruitment to telomeres in PARP1 proficient and deficient cells. Aim 3 will test PARP1 roles in telomere preservation upon replication stress induced with aphidicholin or G-quadruplex ligands. We will examine the recruitment of PARP1 to telomeres, and replication progression at telomeres in PARP1 proficient and deficient cells using the established single molecule analysis of replicated DNA (SMARD) assay. Completion of this project will uncover new interactions and relationships between PARP1 and the telomere specific proteins in preserving telomere integrity after DNA damage and replication stress. In the long term, this work will have important implications for the development of new cancer therapeutic strategies that target PARP1 functions in telomere maintenance.

解读PARP1活性在端粒完整性中的作用机制 本项目的目标是确定聚(ADP-核糖)聚合酶1(PARP1)在端粒保存中的作用 在正常情况下和在环境遗传毒性暴露后。我在PARP领域的深厚背景 而我正在进行的端粒生物学培训，使我处于一个独特的位置，可以成功地完成这项工作 项目。从长远来看，这个项目将为我建立独立的研究奠定基础 研究多聚磷酸酶在基因组稳定性和人类健康中的作用的计划。端粒由TTAGGG组成 由6个成员的Shelterin蛋白复合体结合的重复阵列，并通过相反的方式延长 干细胞和大多数癌细胞中的转录酶、端粒酶。可能会出现端粒磨损和功能障碍 暴露于各种环境因素，并与衰老相关的疾病和癌症有关。 以前的研究已经发现PARP1与端粒维持有关，但其机制还不是很清楚 明白了。PARP1合成多(ADP-核糖)(PAR)，这一活性对基因组维持至关重要 通过促进DNA修复途径和调节复制应激过程中的DNA复制。中环 该提议的假设是，PARP1通过调节端粒的活性来保持端粒的完整性 端粒保护素蛋白和端粒酶。为了支持这一点，我证明了保护素蛋白以及 端粒酶，与PAR结合。我还观察到PAR结合在体外能刺激端粒酶活性。目标1将 确定PARP1在正常条件下和之后与各种Shelterin蛋白的相互作用 遗传毒性侮辱，以及这些相互作用在端粒DNA修复中的作用。我们将测试PAR化 并将确定它们与PAR的亲和力。会导致DNA氧化和烷化损伤 分别与H_2O_2和MNNG作用时，8-氧鸟嘌呤将通过创新的 瞄准系统。我们将跟踪DNA损伤后PARP1在端粒上的募集，并将测量DNA PARP1熟练和缺陷细胞的修复率和端粒畸变率。AIM 2将研究PARP1的角色 在调节端粒酶方面。我们将研究PAR与报告的假定PAR结合基序(PBM)在 HTERT亚基，并将检测这些PBM突变对端粒酶活性的影响。我们会监控 表达野生型和PBM突变型hTERT的细胞的端粒长度变化和端粒酶的募集 到PARP1成熟细胞和缺陷细胞中的端粒。AIM 3将测试PARP1在端粒保存中的作用 在用Ahidicholin或G-四链配体诱导复制应激时。我们会研究招聘 PARP1到端粒，以及PARP1熟练和缺陷细胞中端粒的复制进展 建立了单分子复制DNA分析(SMARD)方法。该项目的完成将揭开 PARP1与端粒特异蛋白在保存端粒中的新相互作用和关系 DNA损伤和复制应激后的完整性。从长远来看，这项工作将产生重要影响。 开发以端粒PARP1功能为靶点的新的癌症治疗策略 维修。