Agarwal, A and Muniyappa, K (2024) Mycobacterium smegmatis putative Holliday junction resolvases RuvC and RuvX play complementary roles in the processing of branched DNA structures. In: Journal of Biological Chemistry, 300 (10).
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Abstract
In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination, and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the Escherichia coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3� and 5� flap DNA, splayed-arm DNA and dsDNA with 3� and 5� overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and ssDNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis. © 2024 The Authors
| Item Type: | Journal Article |
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| Publication: | Journal of Biological Chemistry |
| Publisher: | American Society for Biochemistry and Molecular Biology Inc. |
| Additional Information: | The copyright for this article belongs to the author. |
| Keywords: | Gene encoding; Gluing; Listeria, Branched DNA; Complementarity role; DNA cleavage; DNA structure; Holliday junctions; Junction-specific dnase; Mycobacteria; Mycobacterium smegmatis; Ruvc; Ruvx/yqgf, Escherichia coli, crossover junction endodeoxyribonuclease; crossover junction endodeoxyribonuclease RuvC; DNA ligase; genomic DNA; mycobacterium tuberculosis RuvX; unclassified drug, Article; bacterial strain; bioinformatics; Deinococcus radiodurans; denaturation; DNA annealing; DNA binding; DNA cleavage; DNA structure; electrophoresis; Escherichia coli; extension gene; gel filtration; gel filtration chromatography; gene expression; genome-wide association study; homologous recombination; immunohistochemistry; multiple sequence alignment; Mycobacterium bovis; Mycobacterium leprae; Mycobacterium smegmatis; Mycobacterium tuberculosis; Neisseria gonorrhoeae; overlapping gene; point mutation; recombination repair; Sanger sequencing; sequence alignment; site directed mutagenesis; size exclusion chromatography; Streptomyces griseus; Western blotting |
| Department/Centre: | Division of Biological Sciences > Biochemistry |
| Date Deposited: | 19 Oct 2024 06:53 |
| Last Modified: | 19 Oct 2024 06:53 |
| URI: | http://eprints.iisc.ac.in/id/eprint/86477 |
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