Abstract
Topoisomerases are crucial enzymes in genome maintenance that modulate the topological changes during DNA metabolism. Deinococcus radiodurans, a Gram-positive bacterium is characterized by its resistance to many abiotic stresses including gamma radiation. Its multipartite genome encodes both type I and type II topoisomerases. Time-lapse studies using fluorescently tagged topoisomerase IB (drTopoIB-RFP) and DNA gyrase (GyrA-RFP) were performed to check the dynamics and localization with respect to DNA repair and cell division under normal and post-irradiation growth conditions. Results suggested that TopoIB and DNA gyrase are mostly found on nucleoid, highly dynamic, and show growth phase-dependent subcellular localization. The drTopoIB-RFP was also present at peripheral and septum regions but does not co-localize with the cell division protein, drFtsZ. On the other hand, DNA gyrase co-localizes with PprA a pleiotropic protein involved in radioresistance, on the nucleoid during the post-irradiation recovery (PIR). The topoIB mutant was found to be sensitive to hydroxyurea treatment, and showed more accumulation of single-stranded DNA during the PIR, compared to the wild type suggesting its role in DNA replication stress. Together, these results suggest differential localization of drTopoIB-RFP and GyrA-RFP in D. radiodurans and their interaction with PprA protein, emphasizing the functional significance and role in radioresistance.
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Acknowledgements
We sincerely thank Dr. Hema Rajaram, Bhabha Atomic Research Centre, Mumbai, for her suggestions and support while preparing the manuscript. Miss Himani Tewari is grateful to the Department of Atomic Energy, Government of India, for the research fellowship.
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SM performed the experiments, analyzed the results, and prepared the manuscript. HT performed the experiments and manuscript preparation. RC conducted the experiments and manuscript preparation. HSM analyzed the data and manuscript preparation. SK conceived the idea, planned and conducted the experiments, analyzed the data, wrote the manuscript, and communicated for publication.
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Supplementary file1 Fig S1: Growth studies of D. radiodurans cells expressing RFP fusions of TopoIB and DNAgyrA episomally. Different dilutions of exponentially growing cells of D. radiodurans (R1) expressing TopoIB-RFP and DNAgyrA-RFP under unirradiated (UIrr) and post-irradiation (Irr) were spotted on TYG agar plates. Fig S2: Time-lapse microscopy of a population of D. radiodurans cells expressing TopoIB-RFP. Microscopic images were taken in differential inference contrast (DIC) and TRITC (TopoIB-RFP- red) channels. The scale bar is 1 μm. Fig S3: Time-lapse microscopy of a population of D. radiodurans cells expressing DNAgyrA-RFP. Microscopic images were taken in differential inference contrast (DIC) and TRITC (DNAgyrA-RFP-red) channels. The scale bar is 1 μm. Fig S4: Growth studies of D. radiodurans (R1) and topo:nptII mutant (Δtopo) under unirradiated (UIrr), post-irradiated (Irr) conditions, post-hydroxyurea (HU) treatment (HU-UIrr) and post-HU treatment followed by irradiation (HU-Irr). Different dilutions of exponentially growing cells were spotted on TYG agar plates. Fig S5: Microscopic images of D. radiodurans cells expressing drDNAgyrA-RFP after hydroxyurea (HU) treatment. Cells were grown in TYG broth and monitored under the microscope at specific time intervals (t=hrs) post-HU treatment. Fig S6: DNA binding motif analysis of D. radiodurans DNA Gyrase A. Sequence alignment of D. radiodurans DNA Gyrase A C- terminal domain (CTD) with M. tuberculosis DNA Gyrase A CTD was done using PROMALS3D (a). A schematic representation of the conserved DNA binding motif GyrA-box and a unique GyrA-box-I at the GyrA-CTD is given (b). Boundaries of the motifs are marked as a black box for GyrA-box and a red box for GyrA-box-I. (PDF 1088 KB)
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Mishra, S., Tewari, H., Chaudhary, R. et al. Differential cellular localization of DNA gyrase and topoisomerase IB in response to DNA damage in Deinococcus radiodurans. Extremophiles 28, 7 (2024). https://doi.org/10.1007/s00792-023-01323-1
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DOI: https://doi.org/10.1007/s00792-023-01323-1