Mutagenesis and biochemical studies to understand the mechanism of stimulation of the GTPase McrB by the endonuclease McrC

Abstract

Modified cytosine recognition B and C (McrBC) complex belongs to the class of bacterial modification-dependent restriction enzymes that recognize and cleave methylated DNA. McrBC is a heterooligomeric complex of fourteen subunits (twelve subunits of McrB and two of McrC). McrB performs sequence-specific DNA binding and GTP hydrolysis, while McrC harbours the endonuclease domain. The GTPase is essential for the endonucleolytic activity. McrB harbours a AAA+ domain that functions as the GTPase. Unlike most AAA+ proteins that are stimulated by their substrate, the GTPase activity of McrB is stimulated by the partner protein McrC. Though the interaction between McrB and McrC is important for functional activities, the molecular basis of the interaction remains unknown. As part of the research work, we plan to dissect out the interaction between McrB and McrC using site-directed mutagenesis and biochemical studies. Our study shows that four selected arginine point mutant and a loop deletion mutant of McrC have no effect in stimulation of GTPase activity of McrB. Biochemical assays with wild type proteins (McrBΔN and McrC) also indicates that how much McrC can stimulates the basal GTPase activity of McrB.