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.

解解端粒完整性中PARP1活性的机制该项目的目标是定义聚（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之后。我在PARP领域的强大背景以及我正在进行的端粒生物学培训，将我处于成功完成此任务的独特位置项目。从长远来看，该项目将为我建立独立研究奠定基础计划在基因组稳定性和人类健康中调查PARP的角色。端粒由ttaggg组成重复由6-MEND庇护素蛋白复合物绑定的阵列，并通过反向加长干细胞和大多数癌细胞中的转录酶端粒酶。端粒损耗和功能障碍可能出现从暴露于各种环境药物，与与衰老有关的疾病和癌症有关。先前的研究已涉及PARP1在端粒维持中，但机制却很差理解。 PARP1合成聚（ADP-核糖）（PAR），此活动对于维持基因组至关重要通过促进DNA修复途径并调节复制应力期间的DNA复制。中央该提议的假设是，PARP1通过调节活动的活动来维护端粒的完整性端粒庇护素蛋白和端粒酶。为此，我证明了庇护蛋白以及端粒酶，结合到标准杆。我还观察到，PAR结合在体外刺激端粒酶活性。目标1意志在正常条件下和之后定义PARP1与各种庇护素蛋白之间的相互作用遗传毒性损伤以及这些相互作用在端粒DNA修复中的作用。我们将测试庇护素蛋白质，并将定义其对PAR的亲和力。氧化和烷基化DNA损伤将被诱导使用H2O2和MNNG分别使用创新的端粒局部诱导8-氧定位系统。我们将在DNA损伤后跟随PARP1募集到端粒，并测量DNA PARP1熟练且缺乏细胞的维修率和端粒畸变。 AIM 2将检查PARP1角色在调节端粒酶中。我们将检查与报道的PAR 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。我们将监视表达野生型和PBM突变体HTERT和端粒酶募集的细胞的端粒长度改变在PARP1熟练和缺陷的细胞中端粒。 AIM 3将测试端粒保存中的PARP1角色在用蚜虫或G-四链体配体诱导的复制应力后。我们将研究 PARP1至端粒，以及使用PARP1中端粒的复制进展已建立的复制DNA（SMARD）测定的单分子分析。该项目的完成将发现 PARP1与端粒特异性蛋白之间的新相互作用以及保存端粒的关系 DNA损伤和复制应力后的完整性。从长远来看，这项工作将具有重要的含义为了开发针对端粒功能的新癌症治疗策略维护。

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.