Structural Insights into the Assembly and Shape of Type III Restriction-Modification (R-M) EcoP15I Complex by Small-Angle X-ray Scattering

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• 作者：Yogesh K. Gupta¹ ; Lin Yang² ; Siu-Hong Chan³ ; James C. Samuelson³ ; Shuang-yong Xu³ ; Aneel K. Aggarwal¹ ; ^{aneel.aggarwal@mssm.edu}
• 关键词：SAXS ; small-angle X-ray scattering ; R&ndash ; M ; Restriction&ndash ; Modification ; TRD ; target recognition domain ; PDB ; Protein Data Bank ; SV ; sedimentation velocity ; BNL ; Brookhaven National Laboratory
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：20 July, 2012
• 卷：420
• 期：4-5
• 页码：261-268
• 文件大小：1023 K

摘要

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 Mod₂ subcomplex. We show that the Mod₂ subcomplex has a relatively compact shape with a radius of gyration (R_G) of 鈭?#xA0;37.4聽脜 and a maximal dimension of 鈭?#xA0;110聽脜. The holoenzyme adopts an elongated crescent shape with an R_G of 鈭?#xA0;65.3聽脜 and a maximal dimension of 鈭?#xA0;218聽脜. From reconstructed SAXS envelopes, we postulate that Mod₂ 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 鈥渟liding clamp鈥?around the DNA.