Plant Molecular Biology

, Volume 68, Issue 4–5, pp 479–491 | Cite as

Homologous recombination properties of OsRad51, a recombinase from rice

  • Chittela Rajanikant
  • Michael Melzer
  • Basuthkar J. Rao
  • Jayashree K. Sainis
Article

Abstract

cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryzasativa, 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.

Keywords

ATPase DNA binding FRET Renaturation Strand exchange Transmission Electron Microscopy 

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

Notes

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.

Supplementary material

11103_2008_9385_MOESM1_ESM.doc (312 kb)
DOC 309 kb

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Chittela Rajanikant
    • 1
  • Michael Melzer
    • 2
  • Basuthkar J. Rao
    • 3
  • Jayashree K. Sainis
    • 1
  1. 1.Plant Biochemistry Section, Molecular Biology DivisionBhabha Atomic Research CenterMumbaiIndia
  2. 2.Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)GaterslebenGermany
  3. 3.Department of Biological SciencesTata Institute of Fundamental Research, ColabaMumbaiIndia

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