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

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...

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Autores principales:	Vandna Kukshal, Taran Khanam, Deepti Chopra, Nidhi Singh, Sabyasachi Sanyal, Ravishankar Ramachandran
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8f920c67fb9b4df8901930b6b7595849
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spelling	oai:doaj.org-article:8f920c67fb9b4df8901930b6b75958492021-11-18T07:21:07ZM. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.1932-620310.1371/journal.pone.0035702https://doaj.org/article/8f920c67fb9b4df8901930b6b75958492012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22545130/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Vandna KukshalTaran KhanamDeepti ChopraNidhi SinghSabyasachi SanyalRavishankar RamachandranPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 4, p e35702 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Vandna Kukshal Taran Khanam Deepti Chopra Nidhi Singh Sabyasachi Sanyal Ravishankar Ramachandran 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.
format	article
author	Vandna Kukshal Taran Khanam Deepti Chopra Nidhi Singh Sabyasachi Sanyal Ravishankar Ramachandran
author_facet	Vandna Kukshal Taran Khanam Deepti Chopra Nidhi Singh Sabyasachi Sanyal Ravishankar Ramachandran
author_sort	Vandna Kukshal
title	M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.
title_short	M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.
title_full	M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.
title_fullStr	M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.
title_full_unstemmed	M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.
title_sort	m. tuberculosis sliding β-clamp does not interact directly with the nad+-dependent dna ligase.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/8f920c67fb9b4df8901930b6b7595849
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M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase.

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