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

Abstract

N 6-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.

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in Borrelia burgdorferi

“…Modified bases on the template DNA strand (i.e., the antisense strand) may slow the rate of transcription elongation, much like the premise for SMRT sequencing described for DNA polymerase. This has been described for various DNA modifications, including m6A in eukaryotic systems (46,47); however, the effects of m6A modification on prokaryote RNA polymerase kinetics are not known. Nonetheless, it is possible that the overrepresentation of m6A modifications on the sense strand of genes minimizes any potential effects on RNA polymerase kinetics in B. burgdorferi.…”

Biochemical and structural basis for YTH domain of human YTHDC1 binding to methylated adenine in DNA

“…On the other hand, one potential disadvantage of using N6mA (or N4mC) in DNA, compared to 5mC, is that the methylated amino group of N6mA (or N4mC) is involved in normal hydrogen-bonding for Watson-Crick base pairing, while the cytosine C5 position is not. Indeed, incorporation of N6mA into a DNA template causes RNA polymerase Pol II pausing, associated with the lower stability and slower kinetics of base pairing, and the stability of N6mA:U base pair is weakened ( 32 ). Similar site-specific pausing at N6mA, by a modified phage DNA polymerase inserting T across from a template N6mA, underlies the ability of PacBio SMRT sequencers to detect methylated adenines ( 33 ), and there is additional evidence for weaker N6mA:T than A:T base pairs ( 34–36 ).…”

Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input

“…The RNAs containing zero, one or two 6mA sites (A-RNA, mA1-RNA and mA2-RNA, respectively) were used as templates for the isothermal replication-scission amplification reaction. As shown in Figure 2F, compared to that on A-RNA, the polymerization efficiency of the KF polymerase decreased by ∼9.1% on the mA1-RNA and further decreased by ∼14.9% on mA2-RNA, demonstrating that the extension of DNA polymerase can be hindered by 6mA (32–34). The changes of polymerization efficiency accumulate for oligos with increasing amounts of base differences.…”