Extended Data Fig. 1: Biochemical characterization of BCDX2-CX3 interaction.
From: BCDX2–CX3 and DX2–CX3 complexes assemble and stabilize RAD51 filaments
a, Domain organization of human RAD51 and its five canonical paralogs. NTD, N-terminal domain. b, Schematic representation of the pull-down experiment. An 8xHis-tag is located at the C-terminus of XRCC2 protein, and BCDX2 complex was immobilized using Ni-NTA beads. For all pull-down experiments, BCDX2 complex contained 2xStrepII-SNAP-tag on RAD51C, CX3 complex had all tags cleaved, RAD51 contained an N-terminal 2xStrepII-SNAP-tag. c, Coomassie-stained SDS-PAGE gel of an 8xHis-BCDX2 pull-down assay incubated either with or without the CX3 complex and either with no nucleotide, or supplemented with 1 mM ADP or 1 mM ATP in all buffers. d, XRCC3 band quantification of pull-down assay shown in (c). Data are presented as mean ± s.d. for n = 3 independent biological replicates. e, Coomassie-stained SDS-PAGE gel of an 8xHis-BCDX2 pull-down assay incubated with the CX3 complex, RAD51 or CX3 and RAD51 in the presence or absence of 100 nt ssDNA. All reactions contained 1 mM ATP. f, XRCC3 and RAD51 band quantification of the assay shown in (e). Data are presented as mean ± s.d. for n = 3 independent biological replicates. g, Surface plasmon resonance (SPR) analysis of immobilized BCDX2, CX3 or BCDX2-CX3 complexes interaction with RAD51 (all tags removed). The inset on the top right shows a dose-response curve, which was used to determine equilibrium binding affinity. All experiments were performed in the presence of 1 mM ATP. MCK – multi-cycle kinetics. h, Size-exclusion chromatography (SEC) reconstitution using Superose 6 3.2/300 (Cytiva) with corresponding SDS-PAGE gels below. 19 nt ssDNA was used as ssDNA substrate. All runs were done in the presence of 1 mM ATP in all buffers. These experiments were repeated at least three times independently with the same result.
