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

描述（由申请方提供）：皮肤黑色素瘤在美国是一个显著且不断增长的公共卫生负担。这种极其致命的疾病是由于位于皮肤表皮底层的黑素细胞中的一系列基因突变而引起的。虽然太阳紫外线辐射是皮肤黑色素瘤的主要原因，但紫外线如何引起黑色素瘤突变的了解甚少。事实上，主要由紫外线直接诱导的环丁烷嘧啶二聚体（CPD）引起的紫外线特征突变在一些与皮肤黑色素瘤有关的突变基因中很常见。然而，UV特征突变占所有黑色素瘤突变的不到10%。因此，由UV间接诱导的DNA损伤，如各种氧化损伤和N-甲基嘌呤（NMPs），和/或由其他尚未鉴定的DNA损伤剂诱导的DNA损伤可能是大多数黑素瘤突变的原因。我们的长期目标是更深入地了解黑色素瘤突变的病因，以及DNA损伤和修复如何参与诱变过程。 系统的核苷酸水平映射的诱导和修复的不同类型的DNA损伤在人类黑色素细胞可以提供宝贵的见解的原因，黑色素瘤突变。这一任务的主要障碍是，目前所有可用的DNA损伤和修复映射的方法缺乏分辨率，灵敏度和/或吞吐量。 这项建议有两个具体目标。在目标1中，我们将开发一种新的非常灵敏的方法，用于在活体人类细胞中进行DNA损伤和修复的高通量核苷酸水平作图。下一代DNA测序技术将用于开发新方法。在目标2中，我们将绘制人类黑素细胞中与黑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NMP，将在来自不同皮肤类型的人的黑素细胞中进行映射。我们计划绘制的基因组区域包括黑色素瘤发生突变的位点及其邻近序列。将系统地评估DNA损伤诱导和修复事件与位点特异性黑色素瘤发生突变的相关性。通过使用可以在数百万或数十亿野生型DNA分子中检测单个突变的技术，将进一步证实所鉴定的病变形成位点特异性突变的趋势。 公共卫生相关性：该项目解决了长期存在的问题，即DNA损伤和修复如何参与黑色素瘤突变的产生。完成拟议的研究将提供宝贵的洞察黑色素瘤的病因，并为预防毁灭性疾病的战略发展非常翔实。

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. PUBLIC HEALTH RELEVANCE: This project addresses the long standing question regarding how DNA damage and repair are implicated in the generation of melanomagenesis mutations. Accomplishment of the proposed studies will offer invaluable insight into the etiological causes of melanoma and be very informative for development of strategies for preventing the devastating disease.