Fig. 4. In vitro DNA ligation assay to assess the functionality of AvLigE protein.
a Graphical scheme representing the workflow of immuno-depletion of AvLigE from A. vaga protein extracts. The rotifers were irradiated at 1 kGy to stimulate the production of AvLigE-B and the proteins were extracted at t72h post-irradiation. After extraction, AvLigE-B was immuno-depleted from the extracts. A non-irradiated control was also added to the analysis (NI). b Single qualitative Western-blot analysis demonstrating the efficiency of AvLigE-B immuno-depletion. See text for details. c Principle of the DNA ligase assay used. A complex of two oligonucleotides is assembled to form a hairpin loop containing a DNA nick to mimick a single-strand break (SSB). A fluorescein moeity is incorporated at the opposite end of the oligonucleotide structure. The complex is then incubated in the presence of increasing concentrations of A. vaga protein extracts, followed by a denaturation of the oligonucleotide structure. In the absence of any ligation activity, the oligonucleotide carrying fluorescein is lost from the wells during the denaturation process. Conversely, in the presence of a ligation activity, the DNA nick is sealed and the fluorescein moiety is kept in the wells after denaturation. The enzyme activity is determined by quantifying the amount of fluorescein retained in the wells as a result of the denaturation step. Figure adapted from Healing et al. 75. Flc, fluorescein. d DNA ligation activities with increasing concentrations of the various A. vaga protein extracts. Data represent the mean ± SD of four technical replicates. Differential activities were measured by comparison of the slopes of the relative absorbance at 450 nm in the linear portions of the curves. Statistical analysis in comparison to NI condition: one-sided Multiple unpaired t test, *p value ≤ 0.05. Exact pvalues for NI-AvLigEB-depl. condition at concentrations from 0.0 to 6.4 µg*10−1/reaction: >0.999999, 0.870033, 0.889157, 0.511038, 0.001045, 0.962556, 0.977686, 0.977686. Exact p values for IR condition at concentrations from 0.0 to 6.4 µg*10−1/reaction: 0.750223, 0.113340, 0.000260, 0.000260, 0.000114, 0.000001, 0.000027, 0.000016. Exact pvalues for IR-AvLigEB-depl. condition at concentrations from 0.0 to 6.4 µg*10−1/reaction: >0.999999, 0.572836, 0.159447, 0.003717, <0.000001, 0.000012, 0.000002, 0.000154. IR, protein extract from irradiated A. vaga rotifers; IR (AvLigE-B depl.), protein extract from irradiated A. vaga rotifers and immuno-depleted for AvLigE-B; NI, protein extract from non-irradiated A. vaga rotifers; NI (AvLigE-B depl.), protein extract from non-irradiated A. vaga rotifers and immuno-depleted for AvLigE-B.