DNA Interstrand Crosslinks, Cancer and Aging

描述(申请人提供)：DNA不断受损并被修复。DNA修复途径被打乱的遗传病揭示了未修复的损伤的后果。人类的修复缺陷会导致严重的癌症风险或严重的过早衰老。因此，癌症的预防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机制。DNA链间交联(ICL)对细胞是一种独特的挑战，因为这两条DNA链都是共价修饰的，不可分离，排除了DNA的转录、复制和简单的无错误切除修复。对于ICL在哺乳动物细胞中修复的机制，以及内源性交联损伤的水平和这些损伤的生理影响，人们知之甚少。这项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。ERCC1-XPF是核苷酸切除、修复DNA单链上的巨大损伤和修复二价ICL所必需的内切酶。小鼠中ERCC1的基因破坏会导致加速衰老，这不能归因于核苷酸切除修复缺陷。我们假设ERCC1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ERCC1小鼠模型来检验这一假设，并揭示未修复的ICL的后果。此外，来自这些动物的细胞将被用来描述LCL修复的步骤。这个项目的具体目标是：目标1是检验未经修复的DNA ICL有助于衰老和致癌的假设。我们在小鼠ERCC1基因组座位上设计了两个新的突变，导致小鼠发育不全。初步数据表明，与ERCC1缺陷小鼠相比，这些小鼠具有相似的但更广泛的孕激素表型。然而，小鼠的无病生存期和寿命与ERCC1-XPF蛋白水平成正比。为了验证我们的假设，我们将用一种能引起ICL的药物来治疗这些小鼠，并将衰老参数与未治疗的动物进行比较。我们预测ERCC1耗竭的小鼠的表型将因药物治疗而恶化，这表明ICL起了因果作用。此外，我们将测量ERCC1中寿命最长的小鼠的衰老参数，包括肿瘤发病率，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是由氧自由基对细胞膜的破坏引起的，可以产生能够使DNA交联的产物。我们假设LPO是自发ICL的来源，有助于ERCC1小鼠的表型。通过营养干预和化学方法诱导ERCC1缺陷症小鼠LPO。我们预测，两者都会加剧小鼠的衰老。这些实验的结果将表明LPO是否可以影响衰老速度，如果是的话，这种影响是否可以通过饮食控制。目的3是建立一种局部诱导DNAICL的方法，并利用该技术确定LCL修复的顺序步骤。ICL的形成效率低下以及导致ICL的药物引起大量其他DNA损伤的事实阻碍了ICL修复的研究。因此，我们建议开发一种技术，利用化学治疗剂补骨脂素的近红外多光子光激活，仅在细胞核的明确区域引入ICL。此方法将使我们能够将ICL特定事件与单个细胞内的其他修复事件区分开来。这项技术将应用于野生型和ICL修复缺陷的ERCC1-/-细胞，以确定哪些蛋白质在LCL损伤的位置上特定组装，以及按什么时间顺序组装。

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.