Next-generation sequencing-based analysis of the effect of N6-methyldeoxyadenosine modification on DNA replication in human cells

Next-Generation Sequencing-Based Analysis of the Roles of DNA Polymerases ν and θ in the Replicative Bypass of 8-Oxo-7,8-dihydroguanine in Human Cells

“…We next combined shuttle vector technology with nextgeneration sequencing (NGS) analysis 26 to examine how the depletion of Pols ν and θ may influence the replicative bypass of 8-oxoG in human cells. To this end, we prepared a doublestranded shuttle vector containing a single 8-oxoG lesion at a specific site using the gapped vector-based strategy (Figures 3A and S2).…”

“…To this end, we prepared a double-stranded shuttle vector containing a single 8-oxoG lesion at a specific site using the gapped vector-based strategy (Figures A and S2). , Notably, we introduced C/C mispaired nucleobases close to the 8-oxoG lesion site for distinguishing the replication products arising from the 8-oxoG-containing and damage-free complementary strands, and the latter can be employed as an internal control. , We subsequently transfected the 8-oxoG-containing plasmid into HEK293T cells as well as the POLN- or POLQ -knockdown cells. After cellular replication, we extracted the progenies of the 8-oxoG-containing vector from cells and then digested them with exonuclease III and DpnI enzymes so as to remove the residual unreplicated plasmid DNA.…”

“…NEBNext Ultra II DNA Library Prep Kit for Illumina was employed to generate the sequencing library as described previously, with minor changes. , The above-mentioned progeny plasmids were PCR-amplified by employing PrimeSTAR HS DNA Polymerase (TakaRa) and a pair of primers spanning 9 DpnI recognition sites and the initial 8-oxoG lesion site. The resulting PCR products were further amplified by nested PCR with sample-specific barcoded primers (Table S2).…”

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

“…Understanding how nucleic acid modifications affect the flow of genetic information may help to understand their biological functions in living cells. − Several studies have explored the effects of 6mdA on in vitro and cellular DNA replication, and the insignificant effects of 6mdA on the efficiency and fidelity of DNA replication in cells are in keeping with its proposed function as an epigenetic regulator. ,− It has been reported that 6mdA may affect DNA transcription indirectly by modulating the binding of transcription factor to its target sequence or changing the chromatin structure in the promoter region. ,,− In addition, 6mdA can directly interact with Saccharomyces cerevisiae RNA polymerase II and induce transcriptional pause in vitro, though further studies are warranted to understand the impact of 6mdA on transcriptional elongation in cells . It has also been found that 1mdA can exhibit strong inhibitory and marginal mutagenic potentials during replication in Escherichia coli or human cells. , The effects of the regioisomeric 1mdA and 6mdA adducts on perturbing the efficiencies and accuracies of DNA transcription, however, have not been systematically examined in human cells.…”

“…We previously developed a shuttle vector- and mass spectrometry-based assay for the measurement of transcriptional alterations induced by site-specific incorporated DNA lesions in vitro and in mammalian cells. ,, Although this method is more effective in generating quantitative measurements than conventional transcriptional lesion bypass and mutagenesis assays, it still involves labor-intensive experimental procedures as different lesion-bearing vectors are required to be transfected into host cells and analyzed separately. ,, Next-generation sequencing (NGS) methods have offered a high-throughput and affordable way to evaluate the cytotoxic and mutagenic potentials of DNA lesions during replication in E. coli and human cells. ,− In this study, we placed a single 1mdA or 6mdA at a defined position on the transcribed strand of nonreplicative plasmids and adapted the NGS-based method to systematically investigate how these methylated DNA adducts perturb DNA transcription in vitro and in human cells.…”