Oxidative stress, telomere damage and Werner syndrome.

描述（由申请人提供）：随着美国人口统计学继续向老年人倾斜，确定正常衰老中发生的身体退化的过程变得越来越重要。对人类衰老的遗传学分析是可能的，并有望对这些过程产生新的见解。导致人类过早衰老的大多数遗传疾病的基因已经被确定。沃纳综合征（WS）就是这样一种疾病。这项提案的总体目标是使用WS的分子分析，以更好地了解正常人群的衰老过程。WS是一种人类遗传性疾病，具有加速衰老的特征。在大多数WS病例中有缺陷的基因编码具有解旋酶和核酸外切酶活性的多肽。多项研究支持WS蛋白在端粒维持中的作用。端粒丢失被预测为反映在复制期间在所有端粒处均匀丢失的DNA的总和，以及以可变频率在个体端粒处发生的零星丢失事件。后一类事件包括由于端粒DNA的损伤而导致的端粒序列的丢失。使用一个简单的抑制集落形成试验，我们已经发现，原代WS成纤维细胞是有缺陷的氧化端粒损伤的修复。基于我们的初步数据和发表的分析氧化应激对正常成纤维细胞端粒序列丢失的影响和研究的作用，在端粒加工的WS蛋白，我们提出了一个模型，其中WS蛋白参与修复损伤的端粒。在目标1中，我们将研究端粒氧化损伤产生的损伤的性质以及细胞识别和响应这种损伤的机制。在目标2中，我们将研究WS蛋白在氧化应激引起的端粒损伤反应中的作用。端粒酶抑制WS成纤维细胞对氧化应激的敏感性的机制将在目的3中确定。目的1-3中的研究将在组织培养中进行;为了确定氧化应激在端粒缺陷相关表型形成中的作用的体内相关性，在目的4中，我们将研究WS解旋酶、端粒酶RNA组分和超氧化物歧化酶2组合缺陷的小鼠。这些研究提供了氧化应激，端粒功能障碍和人类早衰综合征之间的新联系。目前尚不清楚偶发性丢失事件和末端复制效率低下对总体端粒序列丢失率的相对贡献。增加对偶发损失事件和影响其发生的途径的理解对于设计策略以最小化端粒损失至关重要。

DESCRIPTION (provided by applicant): As population demographics in the US continue to skew towards older ages, determining the processes that underlie the physical deterioration that occurs in normal aging becomes of increasing importance. An analysis of the genetics of human aging is possible and promises to yield new insight into these processes. The genes responsible for most genetic disorders of human premature aging have been identified. Werner's syndrome (WS) is one such disorder. The overall aim of this proposal is to use the molecular analysis of WS to better understand the aging process in the normal human population. WS is a human genetic disorder with features suggestive of accelerated aging. The gene defective in most cases of WS encodes a polypeptide with both helicase and exonuclease activities. Multiple studies support a role or roles of the WS protein in telomere maintenance. Telomere loss is predicted to reflect the sum of DNA lost uniformly at all telomeres during replication, and sporadic loss events occurring at individual telomeres with variable frequency. The latter class of events includes loss of telomere sequences due to damage to telomere DNA. Using a simple inhibition of colony formation assay, we have found that primary WS fibroblasts are defective in repair of oxidative telomere damage. Based on our preliminary data and published analyses of the effect of oxidative stress on telomere sequence loss in normal fibroblasts and studies of the role of WS protein in telomere processing, we suggest a model in which the WS protein participates in repair of damage to telomeres. In Aim 1, we will study the nature of the lesions produced by oxidative damage at telomeres and the mechanisms by which the cell recognizes and responds to this damage. In Aim 2, we will study the role of the WS protein in the response to telomere damage caused by oxidative stress. The mechanism by which telomerase suppresses the sensitivity of WS fibroblasts to oxidative stress will be determined in Aim 3. The studies in Aims 1-3 will be performed in tissue culture; to determine the in vivo relevance of the role of oxidative stress in the development of phenotypes related to telomere deficiency, in Aim 4 we will study mice that are deficient in combinations of the WS helicase, telomerase RNA component, and superoxide dismutase 2. These studies provide a novel link between oxidative stress, telomere dysfunction and a human progeroid syndrome. The relative contribution of sporadic loss events and end-replication inefficiency to rates of overall telomere sequence loss is currently unknown. Increased understanding of sporadic loss events and the pathways influencing their occurrence is of critical importance in devising strategies to minimize telomere loss.