DNA Interstrand Crosslinks, Cancer and Aging

描述（由申请人提供）：DNA不断受损和修复。未修复的损伤的后果由遗传疾病揭示，其中DNA修复途径被破坏。人类的修复缺陷可能导致癌症或戏剧性过早衰老的严重风险。因此，预防癌症和与年龄有关的发病率取决于确定DNA损伤的来源和损伤修复的机制。DNA链间交联（ICL）对细胞提出了独特的挑战，因为两条DNA链都被共价修饰且不可分离，从而排除了DNA的转录、复制和简单的无错误切除修复。ICL在哺乳动物细胞中修复的机制知之甚少，内源性交联损伤的水平和这些损伤的生理影响也知之甚少。本研究的长期目标是了解未修复的DNA ICL的生物学影响以及细胞修复ICL损伤的机制。Ercc 1-Xpf是一种核酸内切酶，用于核苷酸切除修复DNA一条链上的大块损伤和修复二价ICL。Ercc 1基因在小鼠中的破坏导致加速衰老，这不能归因于核苷酸切除修复缺陷。我们假设Ercc 1缺陷小鼠的表型是由于它们不能修复ICL，因此是内源性ICL的结果。提出的实验将利用Ercc 1小鼠模型来测试这一假设，并揭示未修复的ICLs的后果。此外，来自这些动物的细胞将用于描绘lCL修复的步骤。本项目的具体目标是：目标1是验证未修复的DNA ICLs有助于衰老和致癌的假设。我们在小鼠Ercc 1基因组位点设计了两个新的突变，导致低形态小鼠。初步数据表明，这些小鼠具有类似的，但更广泛的，progeroid表型相比，Ercc 1缺陷小鼠。然而，小鼠的无病期和寿命与Ercc 1-Xpf蛋白水平成比例地延长。为了验证我们的假设，我们将用一种导致ICL的药物治疗这些小鼠，并将衰老参数与未治疗的动物进行比较。我们预测Ercc 1缺失小鼠的表型将因药物治疗而加重，表明ICLs的因果作用。此外，我们将测量年龄参数，包括最长寿的Ercc 1小鼠的肿瘤发生率，以确定自发ICL对癌症的贡献。目的2是确定脂质过氧化（LPO）是否促进ICL修复缺陷小鼠的衰老。LPO是由氧自由基对膜的损伤引起的，并且可以产生能够交联DNA的产物。我们假设LPO是促成Ercc 1小鼠表型的自发ICL的来源。将通过化学方法和营养干预在Ercc 1亚型小鼠中诱导LPO。我们预测，这两种情况都会加剧小鼠的早衰症。这些实验的结果将表明LPO是否会影响衰老速度，如果是的话，这种影响是否可以通过饮食来控制。目的3是建立一种局部诱导DNA ICL的方法，并利用该技术确定ICL修复的顺序步骤。ICL修复的研究受到ICL形成效率低下以及导致ICL的药物诱导许多其他DNA损伤的事实的阻碍。因此，我们建议开发一种技术，使用近红外多光子光活化的化疗药物peptide，仅在细胞核的明确区域引入ICLs。这种方法将使我们能够区分ICL特异性事件与单个细胞内的其他修复事件。该技术将应用于野生型和ICL修复缺陷Ercc 1-/-细胞，以确定哪些蛋白质在ICL损伤位点特异性组装以及以何种时间顺序组装。

DESCRIPTION (provided by applicant): DNA is continuously damaged and repaired. The consequences of unrepaired damage are revealed by genetic diseases in which DNA repair pathways are disrupted. Repair deficiency in humans can lead to a profound risk of cancer or dramatic premature aging. Prevention of cancer and age-related morbidity are therefore dependent upon identifying the sources of DNA damage and mechanisms of damage repair. DNA interstrand crosslinks (ICLs) present a unique challenge to cells because both DNA strands are covalently modified and inseparable, precluding transcription, replication and simple error-free excision repair of the DNA. The mechanism by which ICLs are repaired in mammalian cells is poorly understood, as are levels of endogenous crosslink damage and the physiological impact of these lesions. The long term objectives of this research are to understand the biological impact of unrepaired DNA ICLs and the mechanism by which cells repair ICL damage. Ercc1-Xpf is an endonuclease required for nucleotide excision repair of bulky lesions on one strand of DNA and the repair of bivalent ICLs. Genetic disruption of Ercc1 in the mouse causes accelerated aging, which cannot be attributed to defective nucleotide excision repair. We hypothesize that the phenotype of the Ercc1-deficient mice is due to their inability to repair ICLs, and therefore the consequence of endogenous ICLs. Experiments proposed will utilize Ercc1 mouse models to test this hypothesis and to reveal the consequence of unrepaired ICLs. In addition, cells derived from these animals will be used to delineate the steps of lCL repair. The specific aims of this project are: Aim 1 is to test the hypothesis that unrepaired DNA ICLs contribute to aging and carcinogenesis. We engineered two novel mutations in the mouse Ercc1 genomic locus that result in hypomorphic mice. Preliminary data indicates that these mice have a similar, but more extensive, progeroid phenotype compared to the Ercc1-deficient mice. However, the disease-free period and longevity of the mice are extended proportional to Ercc1-Xpf protein levels. To test our hypothesis, we will treat these mice with a drug that causes ICLs and compare aging parameters to untreated animals. We predict that the phenotype of the Ercc1-depleted mice will be exacerbated by drug treatment indicating a causal role for ICLs. In addition, we will measure aging parameters including tumor incidence in the longest-lived of the Ercc1 mice to determine the contribution of spontaneous ICLs to cancer. Aim 2 is to determine if lipid peroxidation (LPO) promotes aging in mice with defective ICL repair. LPO is caused by oxygen radical damage to membranes and can yield products able to crosslink DNA. We hypothesize that LPO is a source of spontaneous ICLs that contribute to the phenotype of the Ercc1 mice. LPO will be induced in Ercc1 hypomorphic mice chemically and through nutritional intervention. We predict that both will exacerbate the progeria of the mice. Results from these experiments will indicate if LPO can affect the rate of aging and if so whether this effect can be controlled through diet. Aim 3 is to develop a method to locally induce DNA ICLs and to use this technique to determine the sequential steps of lCL repair. The study of ICL repair is hampered by the fact that ICLs are formed inefficiently and drugs that cause ICLs induce a host of other DNA damages. Thus we propose to develop a technique to introduce ICLs only in a well-defined region of cell nuclei using near infrared multiphoton photoactivation of the chemotherapeutic agent psoralen. This method will enable us to discriminate ICL-specific events from other repair events within a single cell. The technique will be applied to wild type and ICL repair-defective Ercc1-/- cells to determine what proteins assemble specifically at sites of lCL damage and in what chronological order.