M. tuberculosis Sliding [beta]-Clamp Does Not Interact Directly with the NAD.sup.+ -Dependent DNA Ligase

The sliding [beta]-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 [beta]-clamp (Mtb[beta]-clamp) to 3.0 Å resolution. The protein crystallized in the space grou...

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Bibliographic Details
Published in:PloS one 2012-04, Vol.7 (4), p.e35702
Main Authors: Kukshal, Vandna, Khanam, Taran, Chopra, Deepti, Singh, Nidhi, Sanyal, Sabyasachi, Ramachandran, Ravishankar
Format: Article
Language:English
Subjects:
Crystal structure
DNA
DNA replication
Escherichia coli
Excavating machinery
Health aspects
Ligases
Tuberculosis
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Summary:The sliding [beta]-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 [beta]-clamp (Mtb[beta]-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222.sub.1 with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtb[beta]-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[beta]-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K.sub.d of 300 nM. In bacteria like E. coli, the [beta]-clamp is known to interact with subunits of the clamp loader, NAD.sup.+ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtb[beta]-clamp with MtbLigA and the [gamma]-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtb[beta]-clamp interacts in vitro with the [gamma]-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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0035702