High-throughput high-resolution mapping of DNA damage and repair in human cells.

描述（由申请人提供）：尽管已经表征了许多必需的DNA修复因子，但仍有待完成的巨大任务是理解DNA损伤诱导和修复如何受到不同细胞类型（包括不同类型的癌细胞）基因组中的不同染色质结构顺序、各种表观遗传修饰和众多辅助DNA修复因子的调节。这项任务的主要障碍是目前所有可用的DNA损伤和修复作图方法缺乏分辨率、灵敏度和/或通量。我们的第一个目标是开发一种新的方法，允许在特定的基因组区域或整个基因组中进行高通量高分辨率的DNA损伤和修复作图。我们将通过绘制UV诱导的环丁烷嘧啶二聚体和硫酸二甲酯诱导的N-甲基嘌呤的分布和修复来发展该方法。一旦开发，这种新的方法应适用于映射其他类型的病变。与现有的方法相比，新方法将大大提高通量，灵敏度和定量性，并显着降低劳动强度。这种新方法的成功开发将彻底改变DNA损伤和修复在细胞中的映射方式，并将非常有助于将我们对DNA损伤和修复机制的理解提高到系统水平。 所有的癌细胞在DNA修复的某些方面都有缺陷，这使得它们的基因组异常不稳定。包括黑色素瘤在内的癌细胞中DNA损伤诱导和修复的“特殊性”一直是一个长期存在的谜。许多研究表明，癌细胞中DNA损伤诱导和修复的总体水平不一定与正常细胞不同。此外，最近的高通量测序研究表明，DNA修复基因的突变在散发性（非遗传性）癌症中并不常见。然而，所有的癌细胞都有明显的染色质组织和异常基因表达模式的改变。有趣的是，最近发现位于组成型异染色质（着丝粒和端粒）中的卫星重复序列在癌细胞中大量过表达，这是由于异染色质的整体去抑制。众所周知，染色质结构和基因表达可以影响DNA损伤的诱导和修复。因此，我们假设癌细胞改变了DNA损伤诱导和修复，而不是在整体水平上，但在异常表达的基因和异染色质中存在的基因。我们的第二个目标是检验这个假设。我们将比较人黑素细胞和黑色素瘤细胞的DNA损伤诱导和修复：1）在黑色素瘤细胞中特异性激活或抑制的基因，以及异染色质中存在的卫星重复序列和各种转座子衍生的重复序列。从这些研究中产生的结果可能揭示了为什么黑色素瘤细胞对放射和化学疗法具有如此臭名昭著的抵抗力。 公共卫生相关性：该项目涉及开发一种新方法，该方法允许高通量高分辨率绘制人类细胞中DNA损伤和修复的图谱。开发的新方法将被用来测试的假设，即人类黑色素瘤细胞改变了DNA损伤的诱导和修复，而不是在整体水平，但在异常表达的基因和那些存在于异染色质。完成拟议的研究将是非常有用的，提高我们的理解DNA修复机制的系统水平，并带来新的见解的机制，基因组的不稳定性和耐药性的黑色素瘤细胞的治疗。

DESCRIPTION (provided by applicant): Although many essential DNA repair factors have been characterized, a huge task remains to be accomplished is to understand how DNA damage induction and repair are modulated by different orders of chromatin structures, a variety of epigenetic modifications and numerous accessory DNA repair factors in the genome of different cell types, including different types of cancer cells. The major roadblock to this task i that all currently available methods for DNA damage and repair mapping lack the resolution, sensitivity and/or throughput. Our first goal is to develop a novel method that allows high-throughput high-resolution mapping of DNA damage and repair in either specific genomic regions of interest or the entire genome. We will develop the method by mapping distribution and repair of UV induced cyclobutane pyrimidine dimers and dimethyl sulfate induced N-methylpurines. Once developed this novel method should be adaptable for mapping other types of lesions. Compared to currently existing methods, the novel method will have immensely increased throughput, sensitivity and quantitativeness, and dramatically decreased labor-intensity. Successful development of the novel method will revolutionize the way in which DNA damage and repair are mapped in the cell, and will be extremely useful for raising our understanding of DNA damage and repair mechanisms to the system level. All cancer cells are expected to be defective in some aspect of DNA repair that makes their genome unusually unstable. The 'peculiarity' of DNA damage induction and repair in cancer cells, including melanomas, has been a long-standing enigma. Numerous studies have indicated that the overall levels of DNA damage induction and repair in cancer cells are not necessarily different from those in normal cells. Also, recent high- throughput sequencing studies suggest that mutations in DNA repair genes are infrequent in sporadic (non- hereditary) cancers. However, all cancer cells have visible alteration of gross chromatin organization and abnormal gene expression patterns. Intriguingly, it was found very recently that satellite repeats, which are located in constitutive heterochromatin (centromeres and telomeres), are massively overexpressed in cancer cells, due to global de-repression of heterochromatin. It has been well known that chromatin structure and gene expression can affect DNA damage induction and repair. We therefore hypothesize that cancer cells have altered DNA damage induction and repair not at the overall level but in the genes that are aberrantly expressed and those that are present in heterochromatin. Our second goal is to test this hypothesis. We will compare human melanocytes and melanoma cells for DNA damage induction and repair in 1) the genes that are specifically activated or suppressed in melanoma cells and in the satellite repeats and various transposon- derived repetitive sequences that are present in heterochromatin. The results generated from these studies may shed light on why melanoma cells are so notoriously resistant to radiation- and chemo-therapies. PUBLIC HEALTH RELEVANCE: This project involves the development of a novel method that allows high-throughput high-resolution mapping of DNA damage and repair in human cells. The novel method developed will be utilized to test the hypothesis that human melanoma cells have altered DNA damage induction and repair, not at the overall level but in the genes that are aberrantly expressed and those that are present in heterochromatin. Accomplishment of the proposed studies will be extremely useful for raising our understanding of DNA repair mechanisms to the system level and for bringing about new insights into the mechanisms regarding the genome instability and resistance to therapeutics of melanoma cells.