HDNA2 IN DNA REPLICATION AND TELOMERE STABILITY

描述（由申请方提供）：基因组不稳定性被认为是导致衰老过程的原因。导致这种增加的分子机制仍然不清楚，但最近的工作表明，复制应激和端粒功能障碍引起的DNA损伤有助于这一过程。端粒是位于线性染色体末端的DNA-蛋白质结构，其维持是基因组稳定所必需的。端粒的稳定性是由足够的DNA储备和不断增长的相关蛋白质清单来保证的。TRF 1、TRF 2、RAP 1、TIN 2、TPP 1和POT 1这六种蛋白质形成核心复合物，其组成性地存在于端粒处并且是端粒维持所需的。人们曾经认为Shelterin复合物的功能之一是排除端粒中的复制和修复蛋白。然而，现在越来越多的人认识到Shelterin组分在调节许多DNA复制和修复蛋白的活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中发挥的重要性。目前的挑战是阐明Shelterin复合物如何在分子水平上实现这一功能。这项建议的重点是一个这样的DNA复制和修复蛋白，人类Dna 2（hDna 2），这已经牵连冈崎片段加工，DNA修复，和端粒稳定性在酵母中。在这里，我们展示了几个意想不到的发现，强调了围绕hDNA 2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hDna 2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hDna 2仅限于线粒体（68）。然而，我们还发现hDna 2 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TRF 2相互作用; 5）其消耗导致特征在于姐妹染色单体交换增加的超重组表型;和6）其消耗导致端粒缩短。我们假设hDna 2在细胞核内的DNA复制中起作用，在那里它对于高保真DNA复制、DNA修复和端粒稳定性是重要的。本文提出的实验将描述hDna 2的生物化学活性，其确保高保真DNA复制并通过确定这些活性是否为忠实端粒复制和/或加帽所需来影响端粒稳定性。 公共卫生相关性：正常人类细胞内的染色体不断受到诱导DNA损伤的试剂的攻击。此外，损伤也可能来自细胞DNA复制的正常DNA复制过程。如果不修复，这种DNA损伤将导致突变和染色体不稳定，这是癌细胞的定义特征。由于染色体的完整性至关重要，许多蛋白质已经进化到在测量DNA和纠正出现的任何突变方面发挥互补作用。当这些蛋白质发生突变破坏其活性时，DNA损伤持续存在，突变累积。鉴于维持DNA完整性的重要性，许多研究小组专注于定义负责纠正DNA突变和维持稳定染色体的机制。染色体末端（称为端粒）的维持是对染色体稳定性具有深远影响的一个因素。事实上，端粒稳定性的丧失导致染色体融合-断裂循环，从而驱动染色体不稳定性。在这里，我们研究Dna 2，一种已知影响酵母基因组稳定性的蛋白质。我们发现，hDna 2定位于细胞的“发电站”（即线粒体），在那里它参与DNA复制和修复。此外，我们表明，hDna 2损失导致染色体不稳定和端粒缩短。由于端粒缩短与人类癌细胞的标志基因组不稳定性有关，因此了解hDna 2在DNA和端粒稳定性中的作用至关重要。在本申请中提出的工作将使我们能够定义hDna 2如何有助于端粒稳定性。

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. PUBLIC HEALTH RELEVANCE: Chromosomes within a normal human cell are under constant attack from agents that induce DNA damage. In addition, damage can also arise from the normal DNA replication process where the cellular DNA is copied. If left unrepaired, this DNA damage will lead to mutations and chromosome instability, which are defining characteristics of cancer cells. Because chromosomal integrity is of critical importance, numerous proteins have evolved to play complementary roles in surveying the DNA and correcting any mutations that arise. When these proteins undergo mutations that corrupt their activity, DNA damage persists and mutations accumulate. Given the importance of maintaining DNA integrity, many groups focus on defining mechanisms responsible for correcting DNA mutations and maintaining stable chromosomes. Maintenance of the chromosome ends (referred to as telomeres) is one factor that has a profound impact on chromosome stability. Indeed, loss of telomere stability leads to chromosomal fusion-breakage cycles that drive chromosome instability. Here, we study Dna2, a protein known to influence genomic stability in yeast. We show that hDna2 localizes to the "powerhouse" of the cell (i.e. the mitochondria) where it participates in DNA replication and repair. In addition, we show that hDna2 loss leads to chromosome instability and telomere shortening. Because telomere shortening has been linked to genomic instability, a hallmark of human cancer cells, understanding hDna2's role in DNA and telomere stability is paramount. The work proposed in this application will allow us to define how hDna2 contributes to telomere stability.