title: Structural insights into the assembly and shape of Type III restriction-modification (R-M) EcoP15I complex by small-angle X-ray scattering.
abstract: EcoP15I is the prototype of the Type III restriction enzyme family, composed of two modification (Mod) subunits to which two (or one) restriction (Res) subunits are then added. The Mod subunits are responsible for DNA recognition and methylation, while the Res subunits are responsible for ATP hydrolysis and cleavage. Despite extensive biochemical and genetic studies, there is still no structural information on Type III restriction enzymes. We present here small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analysis of the EcoP15I holoenzyme and the Mod(2) subcomplex. We show that the Mod(2) subcomplex has a relatively compact shape with a radius of gyration (R(G)) of ∼37.4 Å and a maximal dimension of ∼110 Å. The holoenzyme adopts an elongated crescent shape with an R(G) of ∼65.3 Å and a maximal dimension of ∼218 Å. From reconstructed SAXS envelopes, we postulate that Mod(2) is likely docked in the middle of the holoenzyme with a Res subunit at each end. We discuss the implications of our model for EcoP15I action, whereby the Res subunits may come together and form a "sliding clamp" around the DNA.
authors.affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA.
authors.forename: Yogesh K
authors.initials: YK
authors.lastname: Gupta
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.forename: Siu-Hong
authors.initials: SH
authors.lastname: Chan
authors.forename: James C
authors.initials: JC
authors.lastname: Samuelson
authors.forename: Shuang-yong
authors.initials: SY
authors.lastname: Xu
authors.forename: Aneel K
authors.initials: AK
authors.lastname: Aggarwal
mesh_terms: D004247:DNA / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D021122:Protein Subunits; D053838:Scattering, Small Angle; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation; D014965:X-Rays
chemical_list: D020035:Holoenzymes; D021122:Protein Subunits; D004247:DNA; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)