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Figure 7 | BMC Molecular Biology

Figure 7

From: Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica

Figure 7

DNA-binding and homologous strand transfer activities of rEhRAD51. A. Partially-purified rEhRAD51 was incubated with [γ-32P]dATP labeled ssDNA and interactions were resolved through non-denaturing PAGE. Lane 1, free probe. Lanes 2 to 4, ssDNA incubated with increasing amounts of rEhRAD51 (2.5, 5 and 7.5 μg, respectively); lanes 5 to 7, ssDNA incubated with increasing concentrations of mock purified fraction (2.5, 5 and 7.5 μg) as control. Protein-DNA complexes (CI to CV) are shown by arrowheads. B. Partially purified rEhRAD51 was incubated with [α-32P]dATP labeled dsDNA and interactions were resolved through PAGE. Lane 1, free probe. Lanes 2 to 4, dsDNA incubated with increasing amounts of rEhRAD51 (2.5, 5 and 7.5 μg, respectively); lanes 5 to 7, dsDNA incubated with increasing concentrations of mock purified fraction E. coli elution fraction (2.5, 5 and 7.5 μg) as control. Protein-DNA complexes (CI to CV) are shown by arrowheads. C. D-loop reactions containing 10,000 cpm of [γ-32P]dATP-labeled oligonucleotide, circular dsDNA and 0, 2.5, 5 and 7.5 μg of partially-purified rEhRAD51 (lanes 1 to 4) were incubated at 37°C for 30 min with 2 mM of ATP. Negative controls were performed without homologous dsDNA (lane 5) and with heterologous dsDNA oligonucleotide instead of homologous dsDNA (lane 6), both of them using 7.5 μg of EhRAD51 elution fraction. Reaction products were analyzed by agarose gel electrophoresis, transferred to nylon membranes and visualized through a Phosphor Imager. D. Densitometric analysis of D-loop products obtained in C. Results are representative of two independent experiments.

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