Kinetic Advantages of the Run‐On Oligomer or Filamentation Mechanism of a DNA Cleaving Enzyme

Abstract

Filament or run-on oligomer (ROO) formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that cleaves two types of sites, primary and secondary, but cleaves secondary only in the presence of primary. Primary site DNA activates SgrAI to form ROO with rapid DNA cleavage activity and that can incorporate SgrAI/DNA complexes containing secondary site DNA, thereby inducing their cleavage. Structural methods including x-ray crystallography and cryo-electron microscopy have been used to characterize the three-dimensional structure of the ROO of SgrAI bound to DNA. These structures, combined with kinetic FRET measurements of the DNA cleavage reaction, have been used in global modeling and data fitting to derive microscopic rate constants and investigate cooperativity, growth mechanisms, product trapping and to compare to conventional, non-run on oligomer mechanisms. Mutations which disrupt the ROO were tested in vivo for anti-phage activity. The three-dimensional structure of SgrAI bound to primary site DNA and in the run-on oligomer form shows a left-handed helix with 4 SgrAI/DNA complexes per turn, and that could in principle extend indefinitely. Global model data fitting and the derived rate constants show that association of SgrAI/DNA complexes into the run-on oligomer is rate limiting and is also the feature of the mechanism responsible for sequestering activated DNA cleavage of primary and secondary sites on the same DNA molecule as the activating sequences (a.k.a. the primary site sequences). This analysis also shows that only very limited product trapping occurs, and that the run-on oligomers are not limited to dissociating and growing from only the ends. DNA cleavage was found to be ten-fold faster than ROO dissociation, providing a commitment to the enzyme reaction pathway. Finally, simulations with the models and derived rate constants show that the SgrAI run-on oligomer mechanism is advantageous over the conventional, non-run on oligomer mechanism in both speed and sequestration of activated DNA cleavage, particularly in cleavage of secondary sites, not protected by methylation on the host genome. Mutations which reduce ROO formation completely abolish the anti-phage activity of SgrAI in vivo, showing the critical role of filament formation to biological function. Support or Funding Information This work was supported by NSF MCB-1410355 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.