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: 10094058
• 负责人: Elise Fouquerel
• 金额: $ 24.9万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2022-02-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094058
• 关键词: Address; Adenosine Diphosphate Ribose; Affinity; Aging; Aphidicolin; Binding; Biological Assay; 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; Regulation; Reporting; Research; Role; Signal Transduction; Sister Chromatid Exchange; Somatic Cell; 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; preservation; prevent; programs; protein complex; recruit; repaired; replication stress; response; single molecule; stem cells; 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.

破译 PARP1 活性在端粒完整性中的机制 该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 在正常条件下和环境遗传毒性暴露后。我在 PARP 领域的深厚背景 以及我正在进行的端粒生物学培训，使我处于一个独特的位置来成功完成这项工作 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 计划调查 PARP 在基因组稳定性和人类健康中的作用。端粒由 TTAGGG 组成 重复阵列由 6 元庇护蛋白复合物结合，并通过反向延长 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，并与衰老相关疾病和癌症有关。 先前的研究表明 PARP1 与端粒维持有关，但其机制尚不清楚 明白了。 PARP1 合成聚（ADP-核糖）（PAR），这种活性对于基因组维护至关重要 通过促进 DNA 修复途径并在复制应激期间调节 DNA 复制。中央 该提议的假设是 PARP1 通过调节端粒的活性来保持端粒完整性。 端粒庇护蛋白和端粒酶。为了支持这一点，我证明了庇护蛋白以及 端粒酶，与 PAR 结合。我还观察到 PAR 结合会刺激体外端粒酶活性。目标1将 定义正常条件下和之后 PARP1 与各种庇护蛋白之间的相互作用 基因毒性损伤，以及这些相互作用在端粒 DNA 修复中的作用。我们将测试 PARylation 保护蛋白，并将定义其对 PAR 的亲和力。会诱导氧化和烷化 DNA 损伤 分别用 H2O2 和 MNNG，使用创新的方法在端粒局部诱导 8-氧代鸟嘌呤 瞄准系统。我们将追踪 DNA 损伤后 PARP1 招募到端粒的情况，并测量 DNA PARP1 充足和缺陷细胞的修复率和端粒畸变。目标 2 将检查 PARP1 角色 调节端粒酶。我们将检查 PAR 与报告的推定 PAR 结合基序 (PBM) 的结合 hTERT 亚基，并将通过突变这些 PBM 来检查对端粒酶活性的影响。我们将监控 表达野生型和 PBM 突变体 hTERT 的细胞中端粒长度的变化以及端粒酶的募集 PARP1 充足和缺陷细胞中的端粒。目标 3 将测试 PARP1 在端粒保存中的作用 由阿菲迪胆碱或 G-四联体配体诱导的复制应激。我们将审查招聘 PARP1 到端粒，以及 PARP1 充足和缺陷细胞中端粒的复制进程 建立了复制 DNA 的单分子分析 (SMARD) 测定法。该项目的完成将揭示 PARP1 和端粒特异性蛋白在保护端粒方面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 开发针对端粒中 PARP1 功能的新癌症治疗策略 维护。

项目成果

γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue.

γPNA FRET 对微型探针用于细胞和组织中端粒 DNA 的定量荧光原位杂交。

• 发表时间: 2017-12-02
• 作者: Orenstein, Alexander;Berlyoung, April S;Rastede, Elizabeth E;Pham, Ha H;Fouquerel, Elise;Murphy, Connor T;Leibowitz, Brian J;Yu, Jian;Srivastava, Tumul;Armitage, Bruce A;Opresko, Patricia L
• 通讯作者: Opresko, Patricia L

Functions of ADP-ribose transferases in the maintenance of telomere integrity.

ADP-核糖转移酶在维持端粒完整性中的功能。

• 发表时间: 2022-03-29
• 作者: Muoio, Daniela;Laspata, Natalie;Fouquerel, Elise
• 通讯作者: Fouquerel, Elise