Fig. 4: The role of hORC6 in DNA licensing. | Nature Communications

Fig. 4: The role of hORC6 in DNA licensing.

From: Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

Fig. 4

Specificity of the pre-RC assay was assessed in the presence (WT pre-RC) and absence of hORC6 (hORC6(-)) under (a) low and (b) high salt conditions. c Normalised hMCM2-7 signal in the presence and absence of hORC6 after high salt wash conditions (as in b). Mean plotted; individual data points are marked with black circles. n = 3 independent experiments, error bars represent standard deviation, statistical significance was calculated using paired, two-tailed t test, *P ≤ 0.05 (P = 0.0246). d Pre-RC assays were carried out under low salt conditions in the presence of ATP or ATPγS. e Two-step pre-RC assay to assess the timing of hORC6 function carried out in low salt conditions with ATP or ATPγS. The absence and presence of hORC6 is denoted by (–) and (+) respectively. Lane 1 represents the input for the second part of the reactions, shown in lanes 3–7. All SDS-PAGE gels are representative of three independent biological replicates. f Normalised hMCM2-7 signal following the two-step pre-RC assay under low salt conditions. Lane 4–7 from (e) are quantified. The absence and presence of hORC6 is denoted by (−) and (+) respectively. Mean plotted; individual data points are marked with black circles. n = 3 independent experiments, error bars represent standard deviation, statistical significance was calculated using RM one-way ANOVA with Tukey’s multiple comparisons test, ns—not significant, *P ≤ 0.05. P values as presented on graph from left to right: P = 0.0381, 0.0995, 0.0414, 0.0380. g An in-solution assay was developed to probe double hexamer formation in the presence and absence of hORC6 by negative stain EM. h The resulting analysis of double hexamer formation was determined based on 2D class averages resulting after 2 rounds of 2D classification from samples applied onto grids at the indicated timepoints. Mean plotted, individual data points are marked with black circles. Three independent experiments, error bars represent the standard deviation. Source data are provided as a Source Data file.

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