Abstract:
The sliding b-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis b-clamp (Mtbb-clamp) to 3.0 A° resolution. The
protein crystallized in the space group C2221 with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 A° respectively. Mtbb-clamp
is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains
with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbbclamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent Kd of 300 nM. In bacteria like E. coli, the b-clamp is known to interact with subunits of the clamp loader, NAD+ - dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbb-clamp with MtbLigA and the cclamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments
and also functionally. Intriguingly while Mtbb-clamp interacts in vitro with the c-clamp loader, it does not interact with
MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria.
