Potential Rad54 separation of function mutation highlights unique roles during homologous recombination
Fig 5
Rad54 S816D/S816D has reduced affinity for dsDNA.
(A). Representative 0.5% TBE gel for EMSA with Rad54 and Rad54 S816D/S817D with a 90 bp Atto647N labelled dsDNA. (B). Graph representing the quantification of the EMSE data for Rad54, Rad54 S816A/S817A, Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. The data are fit by a hill equation. The error bars represent the standard error measurement of at least four independent experiments. (C). Graph representing the Kd measurements for individual EMSA experiments for Rad54, Rad54 S816A/S817A, Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. The bar represents the mean, and the error bars represent the standard error measurement of the experiment. (D). Graph representing the amount of ADP produced per minute in an ATP hydrolysis assay for Rad54, Rad54 S816A/S817A, Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. The bar represents the mean, and the error bars represent the standard deviation of four independent experiments. (E). Graph representing the amount of ADP produced per min for Rad54 and Rad54 S816D/S817D. The bar represents the mean, and the error bar represents the standard deviation of 3 independent experiments. (F). Schematic diagram illustrating DNA curtains experiments to test the activity of Rad54 on dsDNA. This image is reproduced through a CC-BY 4.0 license from [95]. (G). Widefield microscope image for GFP-Rad54, GFP-Rad54 S816A/S817A, and GFP-Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. (H). Mean fluorescent decay rate for Rad54 (Blue), Rad54 S816A/S817A (Green), Rad54 S816D/S817D (Orange), Rad54 D525S/D527S/S816D/S817D (Magenta) and Rad54 with ADP (Cyan). The black line gives the photobleaching rate. The shade represents the standard error measurement of the data. (I). Graph representing the koff for GFP-Rad54 (+ATP), GFP-Rad54 S816A/S817A, and GFP-Rad54 S816D/S817D, Rad54 D525S/ D527S/S816D/S817D and GFP-Rad54 (+ADP) from dsDNA. The bars represent the mean, and the error bars the standard error measurement of the data.