Structures, mechanisms, and kinetic advantages of the SgrAI filament forming mechanism.

Abstract

This review documents investigations leading to the unprecedented discovery of filamentation as a mode of enzyme regulation in the type II restriction endonuclease SgrAI. Filamentation is defined here as linear or helical polymerization of a single enzyme as occurs for SgrAI, and has now been shown to occur in many other enzyme systems, including conserved metabolic enzymes. In the case of SgrAI, filamentation activates the DNA cleavage rate by up to 1000-fold and also alters the enzyme's DNA sequence specificity. The investigations began with the observation that SgrAI cleaves two types of recognition sequences, primary and secondary, but cleaves the secondary sequences only when present on the same DNA as at least one primary. DNA cleavage rate measurements showed how the primary sequence is both a substrate and an allosteric effector of SgrAI. Biophysical measurements indicated that the activated form of SgrAI, stimulated by binding to the primary sequence, consisted of varied numbers of the SgrAI bound to DNA. Structural studies revealed the activated state of SgrAI as a left-handed helical filament which stabilizes an altered enzyme conformation, which binds a second divalent cation in the active site. Efforts to determine the mechanism of DNA sequence specificity alteration are ongoing and current models are discussed. Finally, global kinetic modeling of the filament mediated DNA cleavage reaction and simulations of in vivo activity suggest that the filament mechanism evolved to rapidly cleave invading DNA while protecting the Streptomyces host genome.