DNA Interstrand Crosslinks, Cancer and Aging

描述（由申请人提供）：DNA 不断受损和修复。 DNA 修复途径被破坏的遗传疾病揭示了未修复损伤的后果。人类的修复缺陷可能导致罹患癌症或严重过早衰老的巨大风险。因此，预防癌症和与年龄相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) 对细胞提出了独特的挑战，因为两条 DNA 链均经过共价修饰且不可分离，从而阻碍了 DNA 的转录、复制和简单的无差错切除修复。人们对 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以及这些损伤的生理影响也是如此。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 是一种核酸内切酶，用于 DNA 链上大块损伤的核苷酸切除修复和二价 ICL 的修复。小鼠中 Ercc1 的基因破坏会导致加速衰老，但这不能归因于有缺陷的核苷酸切除修复。我们假设 Ercc1 缺陷小鼠的表型是由于它们无法修复 ICL，因此是内源性 ICL 的结果。拟议的实验将利用 Ercc1 小鼠模型来检验这一假设并揭示未修复 ICL 的后果。此外，来自这些动物的细胞将用于描述 ICL 修复的步骤。该项目的具体目标是： 目标 1 是检验未修复的 DNA ICL 会导致衰老和致癌的假设。我们在小鼠 Ercc1 基因组位点中设计了两个新突变，导致小鼠出现低等态。初步数据表明，与 Ercc1 缺陷小鼠相比，这些小鼠具有相似但更广泛的早衰表型。然而，小鼠的无病期和寿命与 Ercc1-Xpf 蛋白水平成正比。为了验证我们的假设，我们将使用引起 ICL 的药物治疗这些小鼠，并将衰老参数与未经治疗的动物进行比较。我们预测药物治疗会加剧 Ercc1 缺失小鼠的表型，这表明 ICL 具有因果作用。此外，我们将测量衰老参数，包括寿命最长的 Ercc1 小鼠的肿瘤发生率，以确定自发 ICL 对癌症的影响。目标 2 是确定脂质过氧化 (LPO) 是否会促进 ICL 修复缺陷小鼠的衰老。 LPO 是由氧自由基对膜造成的损伤引起的，可以产生能够交联 DNA 的产物。我们假设 LPO 是自发 ICL 的来源，有助于 Ercc1 小鼠的表型。将通过化学方法和营养干预在 Ercc1 低效型小鼠中诱导 LPO。我们预测两者都会加剧小鼠的早衰。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，是否可以通过饮食来控制这种影响。目标 3 是开发一种局部诱导 DNA ICL 的方法，并使用该技术确定 ICL 修复的顺序步骤。 ICL 修复的研究因 ICL 形成效率低下以及引起 ICL 的药物会诱发许多其他 DNA 损伤而受到阻碍。因此，我们建议开发一种技术，利用化疗剂补骨脂素的近红外多光子光活化，仅在细胞核的明确区域中引入 ICL。这种方法将使我们能够区分单个细胞内 ICL 特异性事件和其他修复事件。该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 ICL 损伤位点特异性组装以及以什么时间顺序组装。

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.