DNA damage and repair in human melanocytes: relation to melanomagenesis mutations

描述(由申请人提供)：皮肤黑色素瘤在美国是一个重要的且不断增长的公共卫生负担。这种极其致命的疾病是由于皮肤表皮底层的黑素细胞发生了一系列基因突变而产生的。虽然太阳紫外线辐射是皮肤黑色素瘤的主要原因，但紫外线如何导致黑色素瘤突变尚不清楚。事实上，紫外线信号突变主要由紫外线直接诱导的环丁烷嘧啶二聚体(CPD)引起，在一些与皮肤黑色素瘤有关的突变基因中很常见。然而，紫外线特征突变在所有黑色素瘤突变中所占比例不到10%。因此，紫外线间接诱导的DNA损伤，如氧化性损伤和N-甲基嘌呤(NMP)的种类，和/或其他尚未确定的DNA损伤剂诱导的DNA损伤，可能是大多数黑色素瘤发生突变的原因。我们的长期目标是更深入地了解黑色素瘤发生突变的病因，以及DNA损伤和修复如何涉及突变过程。人类黑素细胞中不同类型DNA损伤的诱导和修复的系统核苷酸水平图谱可以为黑色素瘤发生突变的原因提供宝贵的见解。这项任务的主要障碍是目前所有可用的DNA损伤和修复图谱绘制方法都缺乏分辨率、灵敏度和/或吞吐量。这项提议包含两个具体目标。在目标1中，我们将开发一种新的极其灵敏的方法，用于高通量核苷酸水平的DNA损伤和修复的定位。新方法的开发将利用下一代DNA测序技术。在目标2中，我们将定位与黑色素瘤发生突变相关的DNA损伤诱导和修复。各种类型的DNA损伤的诱导和修复，包括CPD、氧化损伤和NMPs，将在不同皮肤类型的人的黑素细胞中得到映射。我们计划绘制的基因组区域包括黑色素瘤发生突变的位置及其邻近序列。DNA损伤诱导和修复事件与特定部位的黑色素瘤发生突变的相关性将被系统地评估。通过使用一种可以在数百万或数十亿野生型DNA分子中检测到单一突变的技术，将进一步证实已识别的病变形成部位特异性突变的倾向。

DESCRIPTION (provided by applicant): Cutaneous melanoma represents a significant and growing public health burden in the USA. This extremely deadly disease arises as a result of the acquisition of a series of genetic mutations in the melanocytes located in the bottom layer of the skin's epidermis. Although solar UV radiation is the primary cause of cutaneous melanoma, how UV causes the melanomagenesis mutations is poorly understood. Indeed, UV signature mutations, which are primarily caused by cyclobutane pyrimidine dimers (CPDs) directly induced by UV, are common in some mutated genes implicated in cutaneous melanoma. However, the UV signature mutations account for less than 10% of all melanomagenesis mutations. Therefore, DNA lesions indirectly induced by UV, such as the varieties of oxidative lesions and N-methylpurines (NMPs), and/or those induced by other as-yet-unidentified DNA damaging agents may be responsible for the majority of the melanomagenesis mutations. Our long term goal is to gain a deeper understanding of the etiological causes of melanomagenesis mutations and how DNA damage and repair are implicated in the mutagenesis process. Systematic nucleotide-level mapping of induction and repair of diverse types of DNA lesions in human melanocytes can offer invaluable insights into the causes of the melanomagenesis mutations. The major roadblock to this task is that all currently available methods for DNA damage and repair mapping lack the resolution, sensitivity and/or throughput. This proposal contains two specific aims. In aim 1, we will develop a novel extremely sensitive method for high-throughput nucleotide-level mapping of DNA damage and repair in living human cells. The next- generation DNA sequencing technologies will be utilized for the development of the novel method. In aim 2, we will map DNA damage induction and repair related to melanomagenesis mutations in human melanocytes. The induction and repair of various types of DNA lesions, including CPDs, oxidative lesions and NMPs, will be mapped in melanocytes from people of different skin types. The genomic regions we plan to map include the sites of melanomagenesis mutations and their neighboring sequences. The correlations of the DNA damage induction and repair events with the site-specific melanomagenesis mutations will be systematically assessed. The tendencies of the identified lesions to form the site-specific mutations will be further confirmed by using a technique that can detect a single mutation among millions or billions of wild type DNA molecules.