Epigenetic DNA Modification N⁶‑Methyladenine Causes Site-Specific RNA Polymerase II Transcriptional Pausing

N⁶-Methyladenine (N⁶-mA or 6 mA) is an epigenetic DNA modification in eukaryotic genomes. In contrast to the well-established roles of 5-methylcytosine for epigenetic regulation of gene expression, the functional roles of N⁶-mA remain elusive. In particular, the impact of N⁶-mA modification of the DNA template on RNA polymerase II (pol II) transcription elongation is not known. In this work, using the Saccharomyces cerevisiae pol II transcriptional elongation system as a model, we investigated the molecular mechanism of pol II recognition and processing of N⁶-mA sites via both biochemical and structural approaches. We found that N⁶-mA causes site-specific pol II pausing/stalling. Structural analysis revealed that while N⁶-mA can reach the +1 template position, the stability of the N⁶-mA and UTP base pairing is compromised. Taken together, we reveal that the presence of the 6-methyl group on adenine reduces incorporation efficiency and promotes backtracking translocation. Our studies with yeast pol II provide molecular insights into understanding the impacts of N⁶-mA on pol II transcription dynamics in different organisms.