Research Paper Volume 3, Issue 5 pp 515—532

Mutations in the BRCT binding site of BRCA1 result in hyper-recombination


Figure 2. K1702M binds CtIP poorly relative to wild-type BRCA1 and results in hyper-recombination. (A) HEK293T cells were co-transfected with plasmids expressing Myc-CtIP, FLAG-BRCA1wt, or empty vector (pcDNA3) where indicated. Total cell lysates were immuno-precipitated with anti-FLAG beads and examined for CtIP binding by western blot analysis. DNA-PKcs was used as a loading control. (B) FLAG-BRCA1wt or K1702M were co-transfected with Myc-CtIP and immuno-precipitated FLAG material was examined for CtIP binding as in A. (C) HCC1937/DR-GFP cells infected with the indicated HD-Ad vectors or left uninfected were analyzed for HRR/GFP events by FACS. Error bars show SEM from three independent experiments (**, P < 0.01; ***, P < 0.001). P = 0.0028 and 0.0004 when wild-type BRCA1 was compared to vector control and K1702M, respectively. P = 0.0001 when vector control was compared to K1702M. (D) Western blot analysis for FLAG-BRCA1 expression in infected HCC1937 cells with DNA-PKcs as loading control.