Homologous recombination properties of OsRad51, a recombinase from rice.
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.
Rajanikant
Chittela
C
Plant Biochemistry Section, Molecular Biology Division, Bhabha Atomic Research Center, Mumbai 400085, India.
Melzer
Michael
M
Rao
Basuthkar J
BJ
Sainis
Jayashree K
JK
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005057:Eukaryotic Cells / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology* / Q000235:genetics*; D011485:Protein Binding; D020673:Protein Renaturation; D051135:Rad51 Recombinase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018744:DNA, Plant; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases