{"database":"biostudies-literature","file_versions":[],"scores":{"citationCount":0,"reanalysisCount":0,"viewCount":46,"searchCount":0},"additional":{"omics_type":["Unknown"],"volume":["7(4)"],"submitter":["Kukshal V"],"pubmed_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."],"journal":["PloS one"],"pagination":["e35702"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC3335792"],"repository":["biostudies-literature"],"pubmed_title":["M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase."],"pmcid":["PMC3335792"],"pubmed_authors":["Kukshal V","Ramachandran R","Singh N","Khanam T","Chopra D","Sanyal S"],"view_count":["46"],"additional_accession":[]},"is_claimable":false,"name":"M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.","description":"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.","dates":{"release":"2012-01-01T00:00:00Z","publication":"2012","modification":"2024-11-11T20:21:47.911Z","creation":"2019-03-26T23:18:35Z"},"accession":"S-EPMC3335792","cross_references":{"pubmed":["22545130"],"doi":["10.1371/journal.pone.0035702"]}}