M. tuberculosis sliding ?-clamp does not interact directly with the NAD+-dependent DNA ligase.

ABSTRACT: The sliding ?-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 ?-clamp (Mtb?-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222(1) with cell-dimensions a?=?72.7, b?=?234.9 & c?=?125.1 Å respectively. Mtb?-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 Mtb?-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K(d) of 300 nM. In bacteria like E. coli, the ?-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 Mtb?-clamp with MtbLigA and the ?-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtb?-clamp interacts in vitro with the ?-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.

SUBMITTER: Kukshal V 

PROVIDER: S-EPMC3335792 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature