Assembly and architecture of the modification-dependent restriction enzyme McrBC

Abstract

Conversion of chemical energy released upon hydrolysis of nucleotides to mechanical action by specialized motors drives many cellular processes. AAA+ NTPase is a prominent family of such motors that power diverse functions, including DNA replication, transcriptional activation, protein degradation or refolding, membrane fusion, microtubule dynamics etc. As a component of the restriction enzyme McrBC, AAA+ motor hydrolyses GTP to drive nucleolytic cleavage of foreign DNA that invade Escherichia coli. McrBC recognizes a 5-methylcytosine preceded by a purine (RmC) as target site, and cleave DNA containing at least two target sites separated by 40 to 3000 bp. It is a multi-subunit complex of two proteins - McrB and McrC. McrB has a DNA-binding and the AAA+ GTPase domain, while McrC is an endonuclease. As part of my PhD project, I carried out biochemical, biophysical and structural studies of the enzyme complex. As a first step, the subunits and their active complex were purified. Size exclusion chromatography coupled to multi-angle light scattering revealed that in presence of guanine nucelotides, McrB oligomerized to a hexamer. On addition of McrC, a 720 KDa tetradecamer of twelve subunits of McrB and two subunits of McrC was formed. The purified tetradecamer was competent to hydrolyse GTP and cleave DNA. The oligomers of a GTPase-competent but DNA-binding deficient mutant of McrB, and its complex with McrC were crystallized. X-ray diffraction studies were carried out and experimental phases determined. In combination with a low-resolution map obtained using electron cryomicroscopy, the architecture of McrBC was deduced allowing us to understand the structural basis of GTPase stimulation of McrB by McrC. Using stopped-flow spectroscopy, I measured apparent binding affinities of the protein for a minimal DNA substrate and observed the effect of the substrate on the kinetics of GTP hydrolysis. Also, the intrinsic tryptophan fluorescence of the protein was probed to gain insights into the assembly of the complex, and the mode of DNA substrate binding. The thesis presents the results from the study of the assembly and architecture of McrBC.