Abstract
cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryza sativa, Indica cultivar group) and cloned in to pET28a expression vector. The protein was over expressed in E. coli BL21 (DE3) and purified. Purified OsRad51 could bind single and double stranded DNA, however it showed higher affinity for single stranded DNA. Transmission Electron Microscopy (TEM) studies of OsRad51–DNA complexes showed that this protein formed ring like structures and bound DNA forming filaments. OsRad51 protein promoted renaturation of complementary single strands in to duplex DNA molecules and also showed ATPase activity, which was stimulated by single strand DNA. Fluorescence resonance energy transfer (FRET) assays revealed that OsRad51 promoted homology dependent renaturation as well as strand exchange reactions. Renaturation activity was ATP dependent; however strand exchange activity was ATP independent. This is the first report on in vitro characterization of Rad51 protein from crop plants.
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Abbreviations
- FRET:
-
Fluorescence resonance energy transfer
- IPTG:
-
Isopropyl thio-galactoside
- MALDI-TOF:
-
Matrix assisted laser desorption and time of flight
- Ni-CAM:
-
Nickel chelating affinity matrix
- ss:
-
Single-stranded
- ds:
-
Double-stranded
- RF:
-
Replicative form
- TEM:
-
Transmission Electron Microscopy
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Acknowledgements
We would like to thank all the members of Structural Cell Biology group for TEM work and Dr. Hans-Peter Mock, Dr. Andrea Matros, Applied Biochemistry group for MALDI –TOF analysis at Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany. Thanks are also due to Professor A. K. Tyagi, University of Delhi, New Delhi, India for providing the rice cDNA library. The International Bureau of Federal Ministry of Education and Research, Germany (BMBF) and the Department of Atomic Energy, India, funded this project under Indo-German Collaborative project IND05/009.
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Rajanikant, C., Melzer, M., Rao, B.J. et al. Homologous recombination properties of OsRad51, a recombinase from rice. Plant Mol Biol 68, 479–491 (2008). https://doi.org/10.1007/s11103-008-9385-6
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DOI: https://doi.org/10.1007/s11103-008-9385-6