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.
