title: Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI.
abstract: DNA methylation-dependent restriction enzymes have many applications in genetic engineering and in the analysis of the epigenetic state of eukaryotic genomes. Nevertheless, high-resolution structures have not yet been reported, and therefore mechanisms of DNA methylation-dependent cleavage are not understood. Here, we present a biochemical analysis and high-resolution DNA co-crystal structure of the N(6)-methyladenine (m6A)-dependent restriction enzyme R.DpnI. Our data show that R.DpnI consists of an N-terminal catalytic PD-(D/E)XK domain and a C-terminal winged helix (wH) domain. Surprisingly, both domains bind DNA in a sequence- and methylation-sensitive manner. The crystal contains R.DpnI with fully methylated target DNA bound to the wH domain, but distant from the catalytic domain. Independent readout of DNA sequence and methylation by the two domains might contribute to R.DpnI specificity or could help the monomeric enzyme to cut the second strand after introducing a nick.
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Siwek
authors.forename: Honorata
authors.initials: H
authors.lastname: Czapinska
authors.forename: Matthias
authors.initials: M
authors.lastname: Bochtler
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Bujnicki
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Skowronek
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D000225:Adenine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C020102:endodeoxyribonuclease DpnI; C000730567:6-methyladenine