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

Louisiana State Univ A&M Col Baton Rouge, Baton Rouge LA

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Abstract

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.