HDNA2 IN DNA REPLICATION AND TELOMERE STABILITY

HDNA2 在 DNA 复制和端粒稳定性中的作用

• 批准号: 8678946
• 负责人: Sheila A Stewart
• 金额: $ 28.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8678946
• 关键词: Age; Aging; Aging-Related Process; Agreement; Biochemical; Biological Assay; Biological Models; Cell Aging; Cell Cycle; Cell Nucleus; Cell physiology; Cells; Characteristics; Chromosomal Instability; Chromosomal Stability; Chromosomes; Cleaved cell; Complex; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA repair protein; Data; Defect; Ensure; Evolution; Excision; Focus Groups; Functional disorder; Gene Mutation; Genetic; Genetic Recombination; Genome; Genome Stability; Genomic DNA; Genomic Instability; Genomics; Homologous Gene; Human; Human Activities; In Vitro; Kinetics; Lead; Left; Link; Maintenance; Mammals; Mitochondria; Mitochondrial DNA; Molecular; Mutation; Nature; Okazaki fragments; Phenocopy; Phenotype; Play; Premature aging syndrome; Process; Proteins; Reducing Agents; Repetitive Sequence; Replication-Associated Process; Reporting; Role; S Phase; Series; Signal Pathway; Simian virus 40; Sister Chromatid Exchange; Staging; Stress; Structure; Surgical Flaps; Surveys; System; TINF2 gene; Telomere Maintenance; Telomere Shortening; Telomere-Binding Proteins; Telomeric Repeat Binding Protein 1; Werner Syndrome; Work; Yeasts; base; cancer cell; homologous recombination; insight; loss of function mutation; normal aging; premature; prevent; protein structure; repaired; research study; senescence; telomere

DESCRIPTION (provided by applicant): Genomic instability is postulated to contribute to the aging process. The molecular mechanisms responsible for this increase remain obscure but recent work suggests that DNA damage arising from replication stress and telomere dysfunction contribute to the process. The telomere is a DNA-protein structure located at the termini of linear chromosomes and its maintenance is required for genome stability. Telomere stability is ensured by the presence of sufficient DNA reserves and a growing list of associated proteins. Six proteins, TRF1, TRF2, RAP1, TIN2, TPP1, and POT1 form a core complex that is constitutively present at the telomere and required for telomere maintenance. It was once thought that one function of the Shelterin complex was to exclude replication and repair proteins from the telomere. However, there is now growing appreciation for the importance the Shelterin components play in modulating the activities of many DNA replication and repair proteins to ensure that they contribute to high fidelity telomere replication and maintenance of a stable telomere structure. The current challenge is to elucidate how the Shelterin complex carries out this function at the molecular level. The focus of this proposal is on one such DNA replication and repair protein, human Dna2 (hDna2), which has been implicated in Okazaki fragment processing, DNA repair, and telomere stability in yeast. Here, we demonstrate several unexpected findings that underscore the complexity that surrounds hDna2 and support our proposal to study this protein in human cells. Specifically, we have shown that hDna2 localizes to the mitochondria and contributes to efficient mitochondrial DNA replication and repair (21). This work is in agreement with a previous report that claimed that hDna2 is restricted to the mitochondria (68). However, we also find that hDna2 1) localizes to the nucleus (21); 2) is found within DNA damage foci; 3) localizes to the telomere; 4) interacts with the telomere binding protein TRF2; 5) its depletion results in a hyper- recombination phenotype characterized by increased sister chromatid exchanges; and 6) its depletion leads to telomere shortening. We hypothesize that hDna2 plays a role in DNA replication within the nucleus where it is important for high fidelity DNA replication, DNA repair and telomere stability. Experiments proposed herein will delineate the biochemical activities of hDna2 that ensure high fidelity DNA replication and that impact telomere stability by determining whether these activities are required for faithful telomeric replication and/or capping.

描述（由申请人提供）：推测基因组不稳定性会导致衰老过程。导致这种增加的分子机制仍然不清楚，但最近的研究表明，复制应激和端粒功能障碍引起的 DNA 损伤促成了这一过程。端粒是位于线性染色体末端的 DNA 蛋白质结构，其维护是基因组稳定性所必需的。足够的 DNA 储备和不断增加的相关蛋白质列表确保了端粒的稳定性。 TRF1、TRF2、RAP1、TIN2、TPP1 和 POT1 六种蛋白质形成核心复合物，该复合物持续存在于端粒上，并且是端粒维持所必需的。人们一度认为 Shelterin 复合物的功能之一是排除端粒中的复制和修复蛋白质。然而，现在人们越来越认识到 Shelterin 成分在调节许多 DNA 复制和修复蛋白的活性方面发挥的重要性，以确保它们有助于高保真端粒复制和维持稳定的端粒结构。当前的挑战是阐明 Shelterin 复合物如何在分子水平上发挥这一功能。该提案的重点是一种 DNA 复制和修复蛋白，即人类 Dna2 (hDna2)，它与冈崎片段加工、DNA 修复和酵母中的端粒稳定性有关。在这里，我们展示了几个意想不到的发现，这些发现强调了 hDna2 的复杂性，并支持我们在人类细胞中研究这种蛋白质的提议。具体来说，我们已经证明 hDna2 定位于线粒体并有助于有效的线粒体 DNA 复制和修复 (21)。这项工作与之前的一份报告一致，该报告声称 hDna2 仅限于线粒体 (68)。然而，我们还发现 hDna2 1) 定位于细胞核 (21)； 2) 在 DNA 损伤灶内发现； 3) 定位于端粒； 4) 与端粒结合蛋白TRF2相互作用； 5)其耗尽导致超重组表型，其特征在于姐妹染色单体交换增加； 6) 它的消耗导致端粒缩短。我们假设 hDna2 在细胞核内的 DNA 复制中发挥作用，对于高保真 DNA 复制、DNA 修复和端粒稳定性非常重要。本文提出的实验将描述 hDna2 的生化活性，通过确定这些活性是否是忠实端粒复制和/或加帽所必需的，hDna2 确保高保真度 DNA 复制并影响端粒稳定性。