DNA alkylation lesions can be induced by environmental methylating agents, chemotherapeutics, and natural methyl donors resident within the cell. Unrepaired DNA alkylation lesions can lead to mutations and increase the risk of cancer. The major class of DNA alkylation lesions, N-methylpurines (NMPs), are repaired by the base excision repair (BER) pathway in human cells. Although many types of NMPs have been identified and the enzymology of BER has been well studied, the genome-wide NMP locations and repair kinetics have not been characterized, due to the lack of a high-efficiency NMP mapping method. We have recently developed a high-throughput method named as NMP-seq (N-methylpurine sequencing), in order to characterize the formation and repair of NMP lesions in different chromatin and sequence contexts across the genome. Our preliminary studies in yeast suggest that NMP-seq is able to map MMS (methyl methanesulfonate)-induced N- 7-methylguanine (7meG) and N-3-methyladenine (3meA) lesions throughout the yeast genome at single nucleotide resolution.
In Aim 1, we will use the yeast NMP-seq data to further analyze how the formation and repair of NMPs are regulated by chromatin, transcription factors, and the cellular transcription activity. Importantly, we will also measure genome-wide mutation profile in MMS-treated yeast, and correlate the mutation data with the NMP lesion formation and repair profile generated by NMP-seq. Comparison of the two high-resolution maps will allow us to explicitly characterize how MMS damage and BER influence mutagenesis in the yeast genome. Alkylating agents are widely used in cancer treatment. However, formation and repair of DNA lesions induced by alkylating drugs are poorly understood.
In Aim 2, we will use NMP-seq to map NMP lesions in normal human fibroblasts, after exposure to the alkylating chemotherapeutic agent temozolomide (TMZ). The genome-wide BER kinetics of TMZ-induced NMP lesions will be analyzed in relation to human chromatin state, nucleosome positioning, and active transcription factor binding sites (TFBS), with the goal of understanding how BER is regulated by different genomic features in human cells. Chemoresistance has been found in numerous cancer patients treated with TMZ. It remains unclear if NMP formation and repair are altered in TMZ-resistant cancer cells. We will use NMP-seq to measure NMP lesions in TMZ-sensitive and the matched TMZ-resistant cancer cells. The genome-wide profile of NMP formation and repair in cancer cells should provide new insights into the mechanisms of TMZ resistance.
The precise locations and repair of DNA alkylation lesions have not been mapped. This proposal will use a novel high-throughput method to measure genome-wide DNA alkylation lesions at single nucleotide resolution. Studies proposed in this application will highlight how chromosomal `landscape' in eukaryotic genomes modulates the formation and repair of DNA alkylation lesions.
|Mao, Peng; Brown, Alexander J; Malc, Ewa P et al. (2017) Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity. Genome Res 27:1674-1684|