Next-Generation Sequencing-Based Analysis of the Effects of N1- and N6-Methyldeoxyadenosine Adducts on DNA Transcription.

Abstract

DNA methylation can occur naturally or be induced by various environmental and chemotherapeutic agents. The regioisomeric N1- and N6-methyldeoxyadenosine (1mdA and 6mdA, respectively) represent an important class of methylated DNA adducts. In this study, we developed a shuttle vector- and next-generation sequencing-based assay to quantitatively assess the effects of 1mdA and 6mdA on the accuracy and efficiency of DNA transcription. Our results revealed that 1mdA can induce multiple types of mutant transcripts and strongly inhibit DNA transcription, whereas 6mdA is a nonmutagenic DNA adduct that can exhibit a subtle but significant inhibitory effect on DNA transcription in vitro and in human cells. Moreover, our results demonstrated that the transcription-coupled nucleotide excision repair pathway is dispensable for the removal of 1mdA and 6mdA from the template DNA strand in human cells. These findings provided new important insights into the functional interplay between DNA methylation modifications and transcription in mammalian cells.