TheMycobacterium tuberculosisgenome possesses homologues of theruvCandyqgFgenes 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 ofruvCandyqgFis induced in response to DNA damage. Protein-DNA interaction assays with purifiedM. tuberculosisRuvC (MtRuvC) and YqgF (MtRuvX) revealed that both associate preferentially with HJ DNA, albeit with differing affinities. Although both MtRuvC and MtRuvX cleaved HJ DNAin 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 MtRuvXD28N 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. coliYqgF is monomeric and exhibits no branched DNA binding or cleavage activity. These results fit with a functional modification of YqgF inM. tuberculosisso that it can act as a dimeric HJ resolvase analogous to that of RuvC.
Mycobacterium tuberculosis genome possesses homologues of the ruvC and ruvX (yqgF) genes that encode putative Holliday junction (HJ) resolvases. However, their enzymatic properties have not been experimentally defined. This work reveals that although both MtRuvC and MtRuvX cleaved HJ, the latter displayed robust HJ resolution activity. Strikingly, we found that disulfide-bond mediated dimerization is essential for MtRuvX activity. This is the first example of a Holliday junction resolvase requiring inter-molecular disulfide-bond formation for biological activity.