Novel Allosteric Properties Exhibited by SgrAI, A Restriction Endonulease Native to Streptomyces Griseus

Abstract

An 8-bp recognition sequence, secondary activity, and allosteric activity for the primary sequence make SgrAI a rather peculiar endonuclease. Recent evidence suggests that SgrAI exists as a dimer in solution in the absence of DNA and is able to bind to one duplex of DNA. However, the molecule mechanisms of secondary site and allosteric activity are poorly understood. Through a series of kinetic and binding assays we have reason to believe that the rate of DNA cleavage by SgrAI is significantly accelerated through the formation of HMWS (high molecular weight species), an aggregate of SgrAI dimers bound to DNA. Native gel electrophoresis suggests that increasing concentrations of PCP (precut primary site), in the presence of excess SgrAI enzyme, increases formation of the HMWS. Furthermore, our kinetic assays show acceleration of DNA cleavage by the addition of PCP in the presence of excess SgrAI. As such, it appears that the HMWS is an activated form of the enzyme. Crystal structures show that the tetramer has an N-terminal region which is domain swapped with another dimer to form a tetramer, which may be a building block of the HMWS. In order to further test this hypothesis, we measured HMWS formation and the stimulation of DNA cleavage by mutated enzymes, P27G/W SgrAI. Both mutants enzymes were designed to disrupt the domain swapped tetramer. Native gel electrophoresis suggests that P27W/G SgrAI enzymes do not form HMWS to the same extent as wild-type SgrAI (wtSgrAI). Additionally, P27W/G SgrAI do not exhibit the same acceleration of DNA cleavge as wtSgrAI, which leads us to believe that formation of the tetramer is necessary for formation of the HMWS. It also further supports the hypothesis that the active conformation of the enzyme can be found in the HMWS. This work is supported by Howard Hughes Medical Institute and NIH, General Medical Sciences.