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

Abstract

The sliding β-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 β-clamp (Mtbβ-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. Mtbβ-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 Mtbβ-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 β-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 Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-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.

• Authors: Vandna Kukshal, Taran Khanam, Deepti Chopra, Nidhi Singh, Sabyasachi Sanyal, Ravishankar Ramachandran
• Journal: PloS one (PLoS One) Vol. 7 Issue 4 Pg. e35702 ( 2012) ISSN: 1932-6203 [Electronic] United States
• PMID: 22545130 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• DNA, Bacterial
• Escherichia coli Proteins
• beta subunit, DNA polymerase III
• DNA Polymerase III
• DNA Ligases
• DNA ligase (NAD)

Topics

• Bacterial Proteins (chemistry, metabolism)
• Crystallography, X-Ray
• DNA Ligases (metabolism)
• DNA Polymerase III (chemistry, metabolism)
• DNA, Bacterial (chemistry, metabolism)
• Escherichia coli (chemistry, metabolism)
• Escherichia coli Proteins (chemistry, metabolism)
• Models, Molecular
• Mycobacterium tuberculosis (chemistry, enzymology, metabolism)
• Protein Multimerization
• Protein Structure, Tertiary