Journal: Molecular and biochemical parasitology
Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase
Figure Lengend Snippet: DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).
Article Snippet: All oligonucleotides indicated as radiolabeled were done so using T4 polynucleotide kinase and [32 P-γ]-ATP as described [ ]. ϕX174 (+) virion ssDNA and ϕX174 replicative form I dsDNA were purchased from New England BioLabs; the ϕX174 replicative form I dsDNA was linearized with Apa LI (New England BioLabs). pBluescript was purified from E. coli using a Plasmid Giga kit (Qiagen).
Techniques: Binding Assay, Activity Assay, Thin Layer Chromatography, Incubation, Polyacrylamide Gel Electrophoresis