Fig. 3: The MEILB2-binding interface is required for BRME1 recruitment to meiotic DSBs.
From: MEILB2-BRME1 forms a V-shaped DNA clamp upon BRCA2-binding in meiotic recombination
a The MEILB2–BRME1 interaction involves BRME1 amino-acids V548, L555 and I562, which are buried in the hydrophobic core of the four-helical bundle (top), and are evolutionarily conserved (bottom). Multiple sequence alignment: Mus musculus (Mm), Homo sapiens (Hs), Sus scrofa (Ss), Physeter macrocephalus (Pm), Phyllostomus discolour (Pd) and Xenopus laevis (Xl). The full alignment is shown in Supplementary Fig. 1b. b Amylose pulldown of His-BRME1MBD with MBP–MEILB2α following recombinant co-expression. MEILB2-binding of BRME1 wildtype (WT) is blocked by the BRME1 V548E L555E I562E (3E) mutation; representative of three independent experiments. This SDS–PAGE shows the amylose elution fractions; all other fractions of the same experiment are shown in Supplementary Fig. 4. Expression of GFP–BRME1FL (full-length, wildtype) and GFP–BRME1FL 3E (full-length, 3E mutant) in mouse spermatocytes by in vivo electroporation; representative of three independent experiments. c Immunoblots of testis extracts following electroporation with GFP–BRME1FL and GFP–BRME1FL 3E, blotted with antibodies against GFP (top) and β-Actin (bottom). d Mouse zygotene (left) and pachytene (right) spermatocytes expressing GFP–BRME1FL and GFP–BRME1FL 3E, stained with anti-SYCP3 antibody (red), anti-GFP antibody (green), and 4,6-diamidino-2-phenylindole (DAPI). Scale bars, 5 μm. Source data are provided as a Source data file.
