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Deciphering the mechanisms of PARP1 activity in telomere integrity

破译 PARP1 活性在端粒完整性中的机制

基本信息

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
项目类别：
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

项目摘要

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PARP 1活性在端粒完整性中的机制本项目的目标是确定聚（ADP-核糖）聚合酶1（PARP 1）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领域的强大背景以及我在端粒生物学方面的持续训练，使我处于一个独特的位置，能够成功地完成这项工作。项目从长远来看，本项目将为我建立一个独立的研究奠定基础研究PARP在基因组稳定性和人类健康中的作用。端粒由TTAGGG组成由6元shelterin蛋白复合物结合的重复序列，并被反向延长干细胞和大多数癌细胞中的端粒酶转录酶。端粒磨损和功能障碍可能会出现暴露于各种环境因素，并与衰老相关的疾病和癌症有关。以前的研究表明PARP 1参与端粒的维持，但其机制尚不清楚。明白PARP 1合成聚（ADP-核糖）（PAR），这种活性对基因组的维持至关重要通过促进DNA修复途径和在复制应激期间调节DNA复制。中央该建议的假设是PARP 1通过调节端粒的活性来保持端粒的完整性。端粒保护蛋白和端粒酶。为了支持这一点，我证明了shelterin蛋白以及端粒酶与PAR结合。我还观察到，PAR结合刺激端粒酶活性在体外。目标1将定义PARP 1和各种shelterin蛋白在正常条件下和基因毒性损伤，以及这些相互作用在端粒DNA修复中的作用。我们将检测 shelterin蛋白，并将确定其对PAR的亲和力。氧化和烷基化的DNA损伤将被诱导分别与H2 O2和MNNG，和8-氧代鸟嘌呤将在端粒局部诱导，使用创新的瞄准系统我们将跟踪PARP 1在DNA损伤后向端粒的募集， PARP 1表达正常和缺乏细胞的修复率和端粒畸变。目标2将研究PARP 1的作用在调节端粒酶方面。我们将检查PAR结合到报告的假定PAR结合基序（PBM）， hTERT亚基，并将通过突变这些PBM来检查对端粒酶活性的影响。我们会监察表达野生型和PBM突变型hTERT的细胞中端粒长度的改变和端粒酶募集 PARP 1表达正常和缺乏的细胞中的端粒。Aim 3将测试PARP 1在端粒保存中的作用在用aphidicholin或G-四链体配体诱导的复制应激后。我们将审查招聘 PARP 1与端粒的关系以及PARP 1表达正常和缺失细胞中端粒处的复制进展。建立了复制DNA单分子分析（SMARD）方法。该项目的完成将揭示 PARP 1与端粒特异性蛋白在端粒保存中的新的相互作用和关系 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用于开发靶向端粒中PARP 1功能的新癌症治疗策略上维护

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

DOI：
10.3390/molecules22122117
发表时间：
2017-12-02
期刊：
Molecules (Basel, Switzerland)
影响因子：
0
作者：
Orenstein A;Berlyoung AS;Rastede EE;Pham HH;Fouquerel E;Murphy CT;Leibowitz BJ;Yu J;Srivastava T;Armitage BA;Opresko PL
通讯作者：
Opresko PL

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