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: 8500390
• 负责人: Sheila A Stewart
• 金额: $ 27.37万
• 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/8500390
• 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 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）：假定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晦涩难懂，但最近的工作表明，复制应力和端粒功能障碍引起的DNA损伤有助于该过程。端粒是位于线性染色体末端的DNA蛋白结构，其维持是基因组稳定性所必需的。通过足够的DNA储备和越来越多的相关蛋白清单来确保端粒稳定性。六种蛋白质，TRF1，TRF2，RAP1，TIN2，TPP1和POT1形成了一种核心复合物，该核心复合物在端粒上存在组成型，并且需要进行端粒维护。曾经认为，庇护素复合物的一个功能是将复制和修复蛋白从端粒排除。但是，现在人们对庇护素组件在调节许多DNA复制和修复蛋白的活性方面发挥的重要性越来越多，以确保它们有助于高保真性端粒复制和维持稳定的端粒结构。当前的挑战是阐明庇护所络合物如何在分子水平上执行此功能。该提案的重点是这样的DNA复制和修复蛋白，即人DNA2（HDNA2），该蛋白已与酵母中的冈扎基碎片加工，DNA修复和端粒稳定性有关。在这里，我们展示了一些意外的发现，这些发现突显了围绕HDNA2的复杂性并支持我们在人类细胞中研究该蛋白质的建议。具体而言，我们已经表明HDNA2定位于线粒体，并有助于有效的线粒体DNA复制和修复（21）。这项工作与先前的报告一致，该报告声称HDNA2仅限于线粒体（68）。但是，我们还发现HDNA2 1）定位于核（21）； 2）在DNA损伤焦点内发现； 3）本地化到端粒； 4）与端粒结合蛋白TRF2相互作用； 5）其耗竭导致过度重组表型，其特征是姐妹染色单体交换增加； 6）它的耗竭导致端粒缩短。我们假设HDNA2在核内的DNA复制中起作用，在该细胞核中对于高保真性DNA复制，DNA修复和端粒稳定性很重要。本文提出的实验将描述HDNA2的生化活性，以确保高保真DNA复制，并通过确定是否需要这些活动才能实现忠实的端粒复制和/或封盖来影响端粒稳定性。