Oxidative stress, telomere damage and Werner syndrome.

描述（由申请人提供）：随着美国人口统计数据继续向老年化倾斜，确定在正常衰老过程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。导致人类早衰的大多数遗传疾病的基因已经被确定。沃纳氏综合征（WS）就是这样一种疾病。本提案的总体目标是利用WS的分子分析来更好地了解正常人群的衰老过程。WS是一种人类遗传疾病，具有加速衰老的特征。在大多数WS病例中，有缺陷的基因编码一种具有解旋酶和外切酶活性的多肽。多项研究支持WS蛋白在端粒维持中的一个或多个作用。预计端粒丢失反映了复制过程中所有端粒DNA均匀丢失的总和，以及个别端粒以不同频率发生的零星丢失事件。后一类事件包括端粒DNA损伤引起的端粒序列丢失。通过简单的集落形成抑制实验，我们发现原代WS成纤维细胞在氧化端粒损伤的修复中存在缺陷。基于我们对氧化应激对正常成纤维细胞端粒序列丢失影响的初步数据和已发表的分析，以及对WS蛋白在端粒加工中的作用的研究，我们提出了一个WS蛋白参与端粒损伤修复的模型。在目标1中，我们将研究端粒氧化损伤产生的病变的性质以及细胞识别和响应这种损伤的机制。在Aim 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敏感性的机制将在Aim 3中确定。Aims 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乏相关表型发展中的体内相关性，在Aim 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组合缺乏的小鼠。这些研究提供了氧化应激、端粒功能障碍和人类类早衰综合征之间的新联系。零星丢失事件和末端复制效率低下对整体端粒序列丢失率的相对贡献目前尚不清楚。增加对零星损失事件和影响其发生的途径的理解对于设计最小化端粒损失的策略至关重要。

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.