Effects Of N1-methyladenine And N~6-methyladenine On DNA Transcription In Vitro And In Human Cells

N1-methyladenine and N~6-methyladenine are an important class of DNA methylation modifications.DNA 6mA is naturally present in the human genome and may play an important biological role as a new type of epigenetic marker.The effects of 1mA and 6mA on DNA transcription in human cells remain largely unknown.In this paper,by employing a shuttle vector strategy in conjugation with next-generation sequencing analysis,we systematically investigated how 1mA and 6mA affect the efficiency and fidelity of DNA transcription in vitro and in human cells.We also examined the potential repair mechanism of 1mA and 6mA.The main research contents are summarized as follows:(1)In vitro transcription assay.The lesion-bearing plasmids containing 1mA or6mA were constructed using p TGFP-T7-Hha10 as the parent vector.The 1mA or6mA-bearing plasmids were premixed with the lesion-free plasmid at equal molar ratios and used as DNA transcription templates.We performed the in vitro transcription assays using T7 RNA polymerase or human RNA polymerase II in He La cell nuclear extract.We found that 1mA exhibited strong inhibitory and modest mutagenic effects on DNA transcription in vitro,though no obvious effect of 6mA on DNA transcription was observed.(2)In vivo transcription assay.We co-transfected the pre-mixed DNA transcription templates into human cells and the transcripts were subjected to next generation sequencing-based analysis.We found that 1mA impeded substantially DNA transcription and induced transcriptional mutagenesis in human cells.On the other hand,6mA did not affect the efficiency and fidelity of DNA transcription in human cells.(3)The potential repair mechanism of 1mA and 6mA.We further investigated whether the CSB,XPA and XPC,which are key players in nucleotide excision repair,are involved in the repair of 1mA and 6mA.Using the next-generation sequencing,we assessed the effects of 1mA and 6mA on DNA transcription in CSB,XPA or XPC deficient cells.We found that the depletion of CSB,XPA or XPC exert no significant effects on the transcriptional efficiency and fidelity of 1mA and 6mA in human cells.These results indicated that nucleotide excision repair has negligible roles in the removal of 1mA and 6mA in human cells.In this paper,we investigated the effects of 1mA and 6mA on DNA transcription and their potential repair mechanisms.We found that 1mA,but not 6mA,block DNA transcription strongly and induce transcriptional mutagenesis in vitro and in human cells.Moreover,we found that 1mA and 6mA are not the efficient substrate for nucleotide excision repair.These findings provide new insights into the functional interplay between DNA methylation modifications and transcription in mammalian cells.