Biochemical and Structural Characterization of Nucleotide Hydrolysis and DNA Binding by McrB – an atypical AAA+ GTPase

Abstract

Methylated Cytosine Restriction B and C (McrBC) is a modification-dependent restriction the enzyme from Escherichia coli K-12. It identifies the DNA recognition sequence RmC and restricts any DNA having at least two RmC sites separated by 40-3000 bp. The enzyme is made of two subunits – McrB, which has a DNA binding domain and a AAA+ (extended ATPases Associated with various cellular Activities) motor that is uniquely employed in hydrolyzing GTP rather than ATP, and the endonuclease McrC. The subunits assemble together to form a functional tetradecameric (12McrB2McrC) complex in the presence of GTP. The DNA binding domain of McrB specifically binds to DNA with the RmC recognition site and feeds it to McrC for endonucleolytic cleavage. McrB thus serves as a model system to understand how ring-like motors, in particular, those involved in DNA metabolism, function. The mechanisms of GTP hydrolysis and DNA binding for nucleolytic cleavage by McrB is unknown. As part of my research project, I plan to carry out the biochemical and structural characterization of McrB and McrBC and their mutants to dissect the mechanistic details of GTP hydrolysis and DNA binding. Towards this, we have generated a family of mutants that differentially affect GTP binding, the oligomeric assembly, and the GTPase activity of McrB. The results suggest that McrB has a canonical arginine-finger, MBR349 and a non-canonical sensor-II, McrBR348. Mutating other residues like MBE298, MBR347 and MBD343 which lie in the vicinity of nucleotide binding pocket affect nucleotide binding as well as oligomerization of McrB. To our surprise, proposed DNA loop binding mutants, McrBR253A and McrBR254A also affected the nucleotide binding which suggests an allosteric regulation of nucleotide binding.