title: Structural basis of the methylation specificity of R.DpnI.
abstract: R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support 'crosstalk' between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity.
authors: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors: Karolina
authors: K
authors: Mierzejewska
authors: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland wsiwek@iimcb.gov.pl.
authors: Wojciech
authors: W
authors: Siwek
authors: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors: Honorata
authors: H
authors: Czapinska
authors: Institute of Biochemistry and Biophysics PAS, Pawinskiego 5a, 02-106 Warsaw, Poland.
authors: Magdalena
authors: M
authors: Kaus-Drobek
authors: Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.
authors: Monika
authors: M
authors: Radlinska
authors: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors: Krzysztof
authors: K
authors: Skowronek
authors: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland.
authors: Janusz M
authors: http://orcid.org/0000-0002-6633-165X
authors: JM
authors: Bujnicki
authors: Institute of Biochemistry and Biophysics PAS, Pawinskiego 5a, 02-106 Warsaw, Poland.
authors: Michal
authors: M
authors: Dadlez
authors: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland Institute of Biochemistry and Biophysics PAS, Pawinskiego 5a, 02-106 Warsaw, Poland mbochtler@iimcb.gov.pl.
authors: Matthias
authors: M
authors: Bochtler
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000737:chemistry; D020029:Base Pairing; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary
chemical_list: D000225:Adenine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C020102:endodeoxyribonuclease DpnI; C000730567:6-methyladenine