Fig. 1: RAD51C ovarian cancer variants are conserved amongst vertebrate species and a subset exhibit reduced protein interactions with its binding partner RAD51D.

a RAD51C ovarian cancer variants are highly conserved amongst vertebrate species. Multiple sequence alignment of human RAD51C with different species including monkey (M. fascicularis), mouse (M. musculus), zebrafish (D. rerio), and African clawed frog (X. tropicalis). The sequences were aligned using Clustal Omega and the conservation is indicated with an asterisk (*) for high conservation, a colon (:) for moderate conservation (conserved within amino acid groups of strongly similar properties), or a period (.) for some conservation (conserved within amino acid groups of weakly similar properties). Mutated amino acid residues are indicated in the gray shaded regions and/or are labeled above the residue with the amino acid change code. RAD51C ATPase domains are indicated with a purple box (Walker A motif) and a gold box (Walker B motif). The nuclear localization sequence (NLS) is shown with a light blue box. b Linear protein schematic of human RAD51C. The ovarian cancer variants analyzed in this study are labeled and shown to scale. c RAD51C ovarian cancer variants in the Walker A and Walker B regions exhibit reduced yeast-three-hybrid (Y3H) interaction with RAD51D. Y3H analysis of pGAD-RAD51C wild-type or the pGAD-RAD51C variant with pGBD-RAD51D was performed. pRS416-RAD51B was used to stabilize RAD51C. Yeast were transformed with the indicated plasmids and plated on selective medium (SC-LEU-TRP-URA-HIS). Growth indicates a Y3H interaction. Y3H interaction-deficient variants are indicated in gray. d Quantification of Y3H results was used to determine the relative growth of each RAD51C variant interaction with RAD51D, and mean growth was plotted with standard deviations. For variants E67K, K84N, T120A, M136I, G162A, M165L, V170G, and L182N, n = 4 biological replicates. For variants N71K, F164L, V166G, and G189R, n = 5. All other variants, n = 3. A RAD51C variant with a Y3H interaction with RAD51D between 50-100% of wild-type is indicated with green, whereas a Y3H interaction between 0–50% of wild-type RAD51C is indicated with red. Source data are provided as a Source Data file. e Western blot analysis of yeast cells transformed with the Y3H deficient RAD51C variants are expressed. RAD51C expression was analyzed using α-RAD51C antibodies and equal protein loading was assessed by Kar2 (α-Kar2). The experiment was performed in triplicate. Uncropped and unprocessed blots are provided as a Source Data file.