3rb9 | pdb_00003rb9
Crystal structure of the M. tuberculosis beta clamp
Structural highlights
Function[DPO3B_MYCTU] DNA polymerase III is a complex, multichain enzyme responsible for most of the replicative synthesis in bacteria. This DNA polymerase also exhibits 3' to 5' exonuclease activity. The beta chain is required for initiation of replication once it is clamped onto DNA, it slides freely (bidirectional and ATP-independent) along duplex DNA (By similarity). Publication Abstract from PubMedThe 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 (Mtbbeta-clamp) to 3.0 A resolution. The protein crystallized in the space group C222(1) with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 A respectively. Mtbbeta-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 Mtbbeta-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 beta-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 Mtbbeta-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 Mtbbeta-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. M. tuberculosis sliding beta-clamp does not interact directly with the NAD+-dependent DNA ligase.,Kukshal V, Khanam T, Chopra D, Singh N, Sanyal S, Ramachandran R PLoS One. 2012;7(4):e35702. Epub 2012 Apr 24. PMID:22545130[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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