Fluorescent Labeling of RAD51C and BCDX2

RAD51^C319S was fluorescently labelled with AF488 or AF555 as described^{47 (link)}. BCDX2 was single and dual (FRET) labelled using the ybbr/Sfp transferase and LPXTG/sortase labelling strategies^{53 (link),55 (link)}. For XRCC2 LPXTG/sortase labelling, Resource Q purified B^ybbrCDX2^TS was mixed with a 10-fold molar excess of AF647 labelled peptide (NH2-C^AF647HHHHHHHHHHLPETGG-COOH), recombinant sortase enzyme and 5 mM MgCl₂, and incubated at 4°C overnight to yield B^ybbrCDX2^AF647. For RAD51B ybbr/Sfp transferase labelling, B^ybbrCDX2^TS was mixed with a 3-fold molar excess of CoA-AF647, Sfp transferase enzyme, 5 mM MgCl₂, and incubated overnight at 4°C to yield B^AF647CDX2. For dual labelling, B^ybbrCDX2^TS, B^ybbr/E144ACDX2^TS or B^ybbrC^E161ADX2^TS were mixed with a 10-fold molar excess of AF647 labelled peptide, a 3-fold molar excess of CoA-AF555, Sfp and sortase enzymes, 5 mM MgCl₂ and incubated at 4°C overnight to yield B^AF555CDX2^AF647, B^AF555/E144AcdX2^AF647 and b^AF555C^E161ADX2^AF647, respectively. All fluorescently labelled proteins were gel filtered on a Superdex 200 Increase (either 10/300 or 3.2/100 GL) column (Cytiva) on an ÄKTA Pure to separate protein from fluorescent peptides/molecules and labelling enzymes.