Protein expression and purification

His₆-tagged WT human DMC1 was overexpressed in the E. coli RecA-deficient BLR strain harboring with pRARE to supply tRNAs for rare codons. For purification of the DMC1 protein³⁸, the clarified cell lysate was subjected to TALON affinity resin (TaKaRa), Source Q column (GE Healthcare), macrohydroxyapatite column (GE Healthcare), and Mono Q 5/50 GL column (GE Healthcare). The DMC1-containing fractions were pooled, concentrated in a Centricon-30 concentrator (Millipore), and stored in small aliquots at −80 °C. The DMC1 variants were expressed and purified as described for the WT protein.

Human RAD51 was expressed in E. coli BLR pRARE strain. The protein purification steps were modified from previously described procedure⁴⁰. Briefly, cell extract was subjected to ammonium sulfate (40% saturation) precipitation and resuspended in buffer A (20 mM K₂HPO₄ pH 7.5, 0.5 mM EDTA, 10% glycerol, 0.01% Igepal, 1 mM 2-mercaptoethanol) supplemented with 2 mM Benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 3 μg/ml of the following protease inhibitors: Aprotinin, Chymostatin, Leupeptin, and Pepstatin A. The RAD51 suspension was then chromatographically fractionated in a Sepharose Q column by applying a 180 ml gradient of 150~660 mM KCl in buffer A. The RAD51 pool was diluted with buffer A and further fractionated in a 1 ml macrohydroxyapatite column using a 90 ml linear gradient of 70~560 mM KH₂PO₄ in buffer A containing 50 mM KCl. The pooled RAD51-containing fraction was diluted and further purified by a 1 ml Source Q column with an 80 ml 235~575 mM KCl gradient in buffer A, and following by a 1 ml Mono Q column with a 45 ml 235~490 mM KCl gradient in buffer A. Finally, the RAD51-containing fractions were pooled, concentrated, and stored at −80 °C. The RAD51 variants were expressed and purified as described for the WT protein. E. coli RecA protein was purchased from New England Biolabs.