title: Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.
abstract: For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.
authors.affiliation: Institute of Medical Virology, Helmut-Ruska-Haus, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin,Germany.
authors.forename: Karol H
authors.initials: KH
authors.lastname: Wyszomirski
authors.forename: Ute
authors.initials: U
authors.lastname: Curth
authors.forename: Jürgen
authors.initials: J
authors.lastname: Alves
authors.forename: Petra
authors.initials: P
authors.lastname: Mackeldanz
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Möncke-Buchner
authors.forename: Mike
authors.initials: M
authors.lastname: Schutkowski
authors.forename: Detlev H
authors.initials: DH
authors.lastname: Krüger
authors.forename: Monika
authors.initials: M
authors.lastname: Reuter
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D004247:DNA; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific; D000251:Adenosine Triphosphatases