Sin Resolvase Catalytic Activity and Oligomerization State are Tightly Coupled

JOURNAL OF MOLECULAR BIOLOGY

卷:404

Article

Mouw, Kent W.¹  Steiner, Andrew M.¹  Ghirlando, Rodolfo⁴  Li, Nan-Sheng¹  Rowland, Sally-J.³  Boocock, Martin R.³  Stark, W. Marshall³  Piccirilli, Joseph A.^1,2  Rice, Phoebe A.¹ 

[1] Univ Chicago, Dept Biochem & Mol Biol, Chicago, IL 60637 USA

[2] Univ Chicago, Dept Chem, Chicago, IL 60637 USA

[3] Univ Glasgow, Fac Biomed & Life Sci, Glasgow G12 8QQ, Lanark, Scotland

[4] NIDDKD, Mol Biol Lab, NIH, Bethesda, MD 20892 USA

关键词: site-specific recombination;    DNA topology;    serine recombinase;    resolvase;    3 '-bridging phosphorothiolate-modified DNA;   

DOI  :  10.1016/j.jmb.2010.08.057

来源: Elsevier

【 摘 要 】

Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3'-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex. (C) 2010 Elsevier Ltd. All rights reserved.

【 授权许可】

Free