HDNA2 IN DNA REPLICATION AND TELOMERE STABILITY

描述(由申请人提供)：基因组的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损伤参与了这一过程。端粒是一种位于线性染色体末端的DNA-蛋白质结构，它的维持是基因组稳定所必需的。端粒的稳定性是由足够的DNA储备和越来越多的相关蛋白质保证的。TRF1、TRF2、RAP1、TIN2、TPP1和POT1六种蛋白质形成了一个核心复合体，它结构性地存在于端粒上，是端粒维持所必需的。人们曾经认为，庇护素复合体的一个功能是排除端粒上的复制和修复蛋白。然而，现在越来越多的人认识到保护素在调节许多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所起的重要作用。目前的挑战是在分子水平上阐明庇护素复合体如何执行这一功能。这项建议的重点是这样一种DNA复制和修复蛋白，人DNA2(HDNA2)，它与酵母中Okazaki片段的处理、DNA修复和端粒稳定性有关。在这里，我们展示了几个意想不到的发现，强调了围绕hDNA2的复杂性，并支持我们在人类细胞中研究这种蛋白质的建议。具体地说，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，我们也发现hDna 2)定位于核(21)；2)位于DNA损伤灶内；3)定位于端粒；4)与端粒结合蛋白TRF2相互作用；5)它的缺失导致以姐妹染色单体交换增加为特征的超重组表型；6)它的缺失导致端粒缩短。我们假设hDNA2在细胞核内的DNA复制中发挥作用，在那里它对高保真DNA复制、DNA修复和端粒稳定是重要的。本文提出的实验将描述hDNA2的生化活性，这些活性确保高保真DNA复制，并通过确定这些活动是否为忠实的端粒复制和/或封顶所必需，来影响端粒稳定性。

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.