title: Mycobacterium tuberculosis RuvX is a Holliday junction resolvase formed by dimerisation of the monomeric YqgF nuclease domain.
abstract: The Mycobacterium tuberculosis genome possesses homologues of the ruvC and yqgF genes that encode putative Holliday junction (HJ) resolvases. However, their gene expression profiles and enzymatic properties have not been experimentally defined. Here we report that expression of ruvC and yqgF is induced in response to DNA damage. Protein-DNA interaction assays with purified M. tuberculosis RuvC (MtRuvC) and YqgF (MtRuvX) revealed that both associate preferentially with HJ DNA, albeit with differing affinities. Although both MtRuvC and MtRuvX cleaved HJ DNA in vitro, the latter displayed robust HJ resolution activity by symmetrically related, paired incisions. MtRuvX showed a higher binding affinity for the HJ structure over other branched recombination and replication intermediates. An MtRuvX(D28N) mutation, eliminating one of the highly conserved catalytic residues in this class of endonucleases, dramatically reduced its ability to cleave HJ DNA. Furthermore, a unique cysteine (C38) fulfils a crucial role in HJ cleavage, consistent with disulfide-bond mediated dimerization being essential for MtRuvX activity. In contrast, E. coli YqgF is monomeric and exhibits no branched DNA binding or cleavage activity. These results fit with a functional modification of YqgF in M. tuberculosis so that it can act as a dimeric HJ resolvase analogous to that of RuvC.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, 560012, India.
authors.forename: Astha
authors.initials: A
authors.lastname: Nautiyal
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, 560012, India.
authors.forename: P Sandhya
authors.initials: PS
authors.lastname: Rani
authors.affiliation: Department of Chemistry, School of Biological and Biomedical Sciences, Biophysical Sciences Institute, University of Durham, DH1 3LE, UK.
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Sharples
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, 560012, India.
authors.forename: K
authors.initials: K
authors.lastname: Muniyappa
mesh_terms: D003545:Cysteine; D004249:DNA Damage; D045566:DNA, Cruciform / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016680:Genome, Bacterial; D045565:Holliday Junction Resolvases / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics* / Q000528:radiation effects; D055503:Protein Multimerization; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D045565:Holliday Junction Resolvases; D003545:Cysteine