Abstract
DNA methylation is an important epigenetic modification in many organisms and can occur on cytosine or adenine. N6-methyladenine (6mA) exists widespreadly in bacterial genomes, which plays a vital role in the bacterial restriction-modification system. Recently, 6mA has also been reported to exist in the genomes of a variety of eukaryotes from unicellular organisms to metazoans. There were controversial reports on whether human N6amt1, which was originally reported as a glutamine MTase for eRF1, is a putative 6mA DNA MTase. We report here the crystal structure of human N6amt1–Trm112 in complex with cofactor SAM. Structural analysis shows that Trm112 binds to a hydrophobic surface of N6amt1 to stabilize its structure but does not directly contribute to substrate binding and catalysis. The active site and potential substrate-binding site of N6amt1 are dominantly negatively charged and thus are unsuitable for DNA binding. The biochemical data confirm that the complex cannot bind DNA and has no MTase activity for DNA, but exhibits activity for the methylation of Gln185 of eRF1. Our structural and biochemical data together demonstrate that N6amt1 is a bona fide protein MTase rather than a DNA MTase.
Highlights
DNA methylation is an important modification which occurs mostly on C5-cytosine (5mC), N4-cytosine (4mC), and N6-adenine (6mA) in both prokaryotes and eukaryotes
Crystallization of the N6amt1–Trm[112] complex in the absence and presence of cofactor S-adenosyl-methionine (SAM) both yielded crystals of the SAM-bound complex, indicating that SAM could be co-purified with the complex
We carried out the structural study of human N6amt1–Trm[112] complex and performed in vitro biochemical studies to investigate the substrate specificity of
Summary
DNA methylation is an important modification which occurs mostly on C5-cytosine (5mC), N4-cytosine (4mC), and N6-adenine (6mA) in both prokaryotes and eukaryotes. Among these modifications, 6mA was initially discovered widespread in bacterial genomes and mainly functions as a part of the restriction-modification system to distinguish the host and foreign pathogenic DNAs in order to protect bacteria against viruses[1,2]. 6mA was initially discovered widespread in bacterial genomes and mainly functions as a part of the restriction-modification system to distinguish the host and foreign pathogenic DNAs in order to protect bacteria against viruses[1,2] This modification was considered to be absent in metazoans. Limited functional studies indicate that DNA 6mA modification is a potential epigenetic mark and may play an important role in gene transcription and chromatin remodeling[12,13]
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