title: High-resolution Digital Mapping of N-Methylpurines in Human Cells Reveals Modulation of Their Induction and Repair by Nearest-neighbor Nucleotides.
abstract: N-Methylpurines (NMPs), including N(7)-methylguanine (7MeG) and N(3)-methyladenine (3MeA), can be induced by environmental methylating agents, chemotherapeutics, and natural cellular methyl donors. In human cells, NMPs are repaired by the multi-step base excision repair pathway initiated by human alkyladenine glycosylase. Repair of NMPs has been shown to be affected by DNA sequence contexts. However, the nature of the sequence contexts has been poorly understood. We developed a sensitive method, LAF-Seq (Lesion-Adjoining Fragment Sequencing), which allows nucleotide-resolution digital mapping of DNA damage and repair in multiple genomic fragments of interest in human cells. We also developed a strategy that allows accurate measurement of the excision kinetics of NMP bases in vitro. We demonstrate that 3MeAs are induced to a much lower level by the SN2 methylating agent dimethyl sulfate and repaired much faster than 7MeGs in human fibroblasts. Induction of 7MeGs by dimethyl sulfate is affected by nearest-neighbor nucleotides, being enhanced at sites neighbored by a G or T on the 3' side, but impaired at sites neighbored by a G on the 5' side. Repair of 7MeGs is also affected by nearest-neighbor nucleotides, being slow if the lesions are between purines, especially Gs, and fast if the lesions are between pyrimidines, especially Ts. Excision of 7MeG bases from the DNA backbone by human alkyladenine glycosylase in vitro is similarly affected by nearest-neighbor nucleotides, suggesting that the effect of nearest-neighbor nucleotides on repair of 7MeGs in the cells is primarily achieved by modulating the initial step of the base excision repair process.
authors.affiliation: From the Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803.
authors.forename: Mingyang
authors.initials: M
authors.lastname: Li
authors.affiliation: From the Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803.
authors.forename: Tengyu
authors.initials: T
authors.lastname: Ko
authors.affiliation: From the Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803 shli@lsu.edu.
authors.forename: Shisheng
authors.initials: S
authors.lastname: Li
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D013463:Sulfuric Acid Esters / Q000494:pharmacology
chemical_list: C580784:N3-methyladenine; D013463:Sulfuric Acid Esters; D006147:Guanine; C008450:7-methylguanine; D000225:Adenine; C007482:dimethyl sulfate
keywords: DNA damage; DNA enzyme; DNA repair; N3-methyladenine; N7-methylguanine; alkyladenine glycosylase; base excision repair (BER); human fibroblast; lesion-adjoining fragment sequencing; mutagenesis; nearest-neighbor nucleotides