Fig. 6: Pathogenic mutation in the OB-folds of BRCA2 abolishes the BRCA2 binding to telomeric G4.
a Recombinant BRCA2OB proteins mutated at D2723H and R2973C, respectively, were subjected to EMSA with telomere probes (left). The SDS-page gel of purified BRCA2OB, BRCA2OB-D2723H, BRCA2OB-R2973C mutants (right). b–d Binding properties of full-length BRCA2 with indicated telomere variant repeats. b Schematic illustration of the assay. EGFP-tagged full-length BRCA2 was immunoprecipitated from NFLAP-BRCA2 HeLa cells10,78 and incubated with labeled probes in the presence of 100 mM KCl. Anti-GFP antibody was employed to pull down NFLAP-BRCA2 from the cell lysate. Unbound probes were washed out and NFLAP-BRCA2-bound probes were eluted and subsequently dot blotted. c Dot blots showing the telomere binding properties of full-length BRCA2. HeLa cell lysate served as negative control. Five percent out of the total labeled probes were loaded as controls. Bar graph indicates the relative binding intensity of full-length BRCA2 to DNA probes (mean ± s.e.m.). The result is from six independent experiments. d The effect of OB-folds mutation in binding to telomere G4. Full-length wild-type and mutant BRCA2-expression constructs, tagged with 2X MBP (Maltose binding Protein), were transfected into 293 T cells. Forty-eight hours later, 293 T cell lysates were subjected to BRCA2 pull-down using amylose resin, then subjected to binding to various telomere G4 variants, including TelG5-G3, and washed. Bound probes were dot blotted as in b and c. Five percent of the probes were blotted for control. The result is the representative of three independent experiments. e Western blot analysis of wild-type and mutant BRCA2, performed simultaneously with d after transfection into 293 T cells to assess the expression level of wild-type vs mutant BRCA2 level. The result was repeated in two independent experiments.
