Novel insights into ATP-Stimulated Cleavage of branched DNA and RNA Substrates through Structure-Guided Studies of the Holliday Junction Resolvase RuvX

Thakur, M and Mohan, D and Singh, AK and Agarwal, A and Gopal, B and Muniyappa, K (2021) Novel insights into ATP-Stimulated Cleavage of branched DNA and RNA Substrates through Structure-Guided Studies of the Holliday Junction Resolvase RuvX. In: Journal of Molecular Biology, 433 (13).

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Abstract

Much of our understanding of the homologous recombination (HR) machinery hinges on studies using Escherichia coli as a model organism. Interestingly enough, studies on the HR machinery in different bacterial species casts doubt on the universality of the E. coli paradigm. The human pathogen Mycobacterium tuberculosis encodes two Holliday junction (HJ)-resolvase paralogues, namely RuvC and RuvX; however, insights into their structural features and functional relevance is still limited. Here, we report on structure-guided functional studies of the M. tuberculosis RuvX HJ resolvase (MtRuvX). The crystalline MtRuvX is a dimer in the asymmetric unit, and each monomer has a RNAse H fold vis-à -vis RuvC-like nucleases. Interestingly, MtRuvX also contains some unique features, including the residues essential for ATP binding/coordination of Mg2+ ions. Indeed, MtRuvX exhibited an intrinsic, robust ATPase activity, which was further accentuated by DNA cofactors. Structure-guided substitutions of single residues at the ATP binding/Mg2+coordination sites while markedly attenuating the ATPase activity completely abrogated HJ cleavage, indicating an unanticipated relationship between ATP hydrolysis and DNA cleavage. However, the affinity of ATPase-deficient mutants for the HJ was not impaired. Contrary to RuvC, MtRuvX exhibits relaxed substrate specificity, cleaving a variety of branched DNA/RNA substrates. Notably, ATP hydrolysis plays a regulatory role, rendering MtRuvX from a canonical HJ resolvase to a DNA/RNA non-sequence specific endonuclease, indicating a link between HJ resolvase and nucleic acid metabolism. These findings provide novel insights into the structure and dual-functional activities of MtRuvX, and suggest that it may play an important role in DNA/RNA metabolism. © 2021 Elsevier Ltd

Item Type:	Journal Article
Publication:	Journal of Molecular Biology
Publisher:	Academic Press
Additional Information:	The copyright for this article belongs to Academic Press
Department/Centre:	Division of Biological Sciences > Biochemistry Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	26 Aug 2021 10:48
Last Modified:	26 Aug 2021 10:48
URI:	http://eprints.iisc.ac.in/id/eprint/69315

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