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

人类细胞 DNA 损伤和修复的高通量高分辨率绘图。

基本信息

批准号：
8514607
负责人：
Shisheng Li
金额：
$ 7.25万
依托单位：
LOUISIANA STATE UNIV A&M COL BATON ROUGE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-20 至 2014-06-30
项目状态：
已结题

项目摘要

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.

描述(申请人提供)：尽管许多基本的DNA修复因子已被确定，但仍有一个巨大的任务有待完成，即了解不同类型细胞(包括不同类型的癌细胞)基因组中不同顺序的染色质结构、各种表观遗传修饰和大量辅助DNA修复因子是如何调节DNA损伤诱导和修复的。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。我们的第一个目标是开发一种新的方法，可以高通量、高分辨率地绘制DNA损伤和修复的图谱，无论是在特定的感兴趣的基因组区域还是整个基因组。我们将通过绘制紫外线诱导的环丁烷、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，这种新的方法应该适用于绘制其他类型的病变。与现有的方法相比，新方法将大大提高吞吐量、灵敏度和定量，并显著降低劳动强度。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定位方式，并将极大地有助于将我们对DNA损伤和修复机制的理解提高到系统水平。所有癌细胞都被认为在DNA修复的某些方面存在缺陷，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DNA损伤诱导和修复的“特殊性”一直是一个谜。大量研究表明，癌细胞的DNA损伤诱导和修复的总体水平与正常细胞不一定不同。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)癌症中很少见。然而，所有癌细胞都有明显的大染色质组织改变和异常的基因表达模式。有趣的是，最近发现，由于异染色质的全球抑制，位于结构性异染色质(着丝粒和端粒)中的卫星重复序列在癌细胞中大量过度表达。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损伤的诱导和修复，不是在整体水平上，而是在异常表达的基因和异染色质中存在的基因中。我们的第二个目标是检验这一假设。我们将比较人类黑素细胞和黑色素瘤细胞在DNA损伤诱导和修复方面的情况：1)在黑色素瘤细胞中特异激活或抑制的基因，以及在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。这些研究产生的结果可能会揭示为什么黑色素瘤细胞对放射和化疗具有如此臭名昭著的抗药性。