Extended Data Fig. 2: Mass photometry (MP) analysis of the RAD52-fork interaction.

a-e. Mutations in the DNA binding sites of RAD52 affect the formation of the double-ring RAD52-fork complex. a. Location of the inner DNA binding site (K152/R153; purple) and the bipartite outer DNA binding site (K102/K133/K169/R173) in one of the monomers (teal ribbon representation) in an undecamerc RAD52 ring. The mutations are mapped on the crystal structure of the RAD52-ssDNA complex (PDB: 5XRZ). Cartoon on the right shows position of the two binding site within the double-ring RAD52 structure. b-e. MP analysis of the macromolecular complexes formed by the mutant forms of RAD52. In all experiments, representative distributions are shown for 100 nM (11xFork; light blue), 220 nM (22xFork; medium blue), and 330 nM (33xFork; dark blue) RAD52^IBD or RAD52^OBD. Lines correspond to fitting of the molecular weights distributions with multiple Gaussians using GrapPad Prism. Vertical dotted lines indicate molecular weights of the RAD52 monomer, decamer and undecamer, respectively. Quantification of the peaks performed in the DiscoverMP software is detailed in the Supplemental Table 2. A heterologous DNA fork with a 30 nt lagging strand gap was assembled using oligonucleotides #2, #3, #4 and #6 listed in the Extended Data Table 1. b. Molecular weight distribution of the RAD52^IBD in solution. Note that the RAD52^IBD forms lower molecular weight complexes than the wild type RAD52. c. Molecular weight distributions of the RAD52^IBD bound to heterologous fork. While binding to the fork is evident from the decrease in the peak corresponding for free fork and a shift of the peak corresponding to the RAD52^IBD single ring, the amounts of double-ring complexes are lower compared to the wild type RAD52. d. Molecular weight distribution of the RAD52^OBD in solution. e. Molecular weight distributions of the RAD52^OBD bound to heterologous fork. Virtually no double rings are observed with this mutant. The shift in the single ring peak relative to the free RAD52^OBD suggests binding to the fork. f-i. RAD52 forms double-undecamer structures on the model fork bound by RPA. f. The MP analysis of the 10 nM DNA fork (grey; the heterologous fork with a 30 nt lagging strand gap), 10 nM RPA (light green) and the fork-RPA complex (dark green). g. The MP analysis of RAD52. h. RAD52 + plus 10 nM fork. i. Fork DNA bound by RPA and RAD52. In all experiments, representative distributions are shown for 100 nM (11xFork; light blue), 220 nM (22xFork; medium blue), and 330 nM (33xFork; dark blue) RAD52. The RAD52-fork-RPA complexes are marked with * in i. See Supplemental Table 3 for quantification. j. Mass photometry experiment showing oligomeric states of 110, 220 and 330 nM RAD52 (from light to dark blue) bound four-way DNA junction substrate. Numbers above each peak indicate the number of RAD52 undecamers in each nucleoprotein complex.