DNA strand exchange activity of rice recombinase OsDmc1 monitored by fluorescence resonance energy transfer and the role of ATP hydrolysis

Abstract

Rad51 and disrupted meiotic cDNA1 (Dmc1) are the two eukaryotic DNA recombinases that participate in homology search and strand exchange reactions during homologous recombination mediated DNA repair. Rad51 expresses in both mitotic and meiotic tissues whereas Dmc1 is confined to meiosis. DNA binding and pairing activities of Oryza sativa disrupted meiotic cDNA1 (OsDmc1) from rice have been reported earlier. In the present study, DNA renaturation and strand exchange activities of OsDmc1 have been studied, in real time and without the steps of deproteinization, using fluorescence resonance energy transfer (FRET). The extent as well as the rate of renaturation is the highest in conditions that contain ATP, but significantly less when ATP is replaced by slowly hydrolysable analogues of ATP, namely adenosine 5'-(β,γ-imido) triphosphate (AMP-PNP) or adenosine 5'-O-(3-thio triphosphate) (ATP-γ-S), where the former was substantially poorer than the latter in facilitating the renaturation function. FRET assay results also revealed OsDmc1 protein concentration dependent strand exchange function, where the activity was the fastest in the presence of ATP, whereas in the absence of a nucleotide cofactor it was several fold (≈15-fold) slower. Interestingly, strand exchange, in reactions where ATP was replaced with AMP-PNP or ATP-γ-S, was somewhat slower than that of even minus nucleotide cofactor control. Notwithstanding the slow rates, the reactions with no nucleotide cofactor or with ATP-analogues did reach the same steady state level as seen in ATP reaction. FRET changes were unaffected by the steps of deproteinization following OsDmc1 reaction, suggesting that the assay results reflected stable events involving exchanges of homologous DNA strands. All these results, put together, suggest that OsDmc1 catalyses homologous renaturation as well as strand exchange events where ATP hydrolysis seems to critically decide the rates of the reaction system. These studies open up new facets of a plant recombinase function in relation to the role of ATP hydrolysis.