title: Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.
abstract: Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N(6)-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Guan-Zheng
authors.initials: GZ
authors.lastname: Luo
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.|Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.|College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University, Wuhan 430072, China.
authors.forename: Xiaocheng
authors.initials: X
authors.lastname: Weng
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Ziyang
authors.initials: Z
authors.lastname: Hao
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Miao
authors.initials: M
authors.lastname: Yu
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Deng
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Jianzhao
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Chuan
authors.initials: C
authors.lastname: He
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002696:Chlamydomonas / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D056890:Eukaryota / Q000378:metabolism*; D016678:Genome / Q000235:genetics; D010407:Penicillium / Q000235:genetics / Q000378:metabolism; D010961:Plasmodium / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D000225:Adenine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C020102:endodeoxyribonuclease DpnI; C000730567:6-methyladenine