The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations -- Data Supplement - HTML Page - 08717Fig6Legend.htslp -- Proceedings of the National Academy of Sciences

Fig. 6.
BRCA1 binds to a discrete region of BACH1. (A) A series of ³⁵S-radiolabeled in vitro-translated BACH1 products were tested for their ability to interact with GST and GST-BRCT (BRCA1 residues 1529-1863). Each binding reaction consisted of GST or GST-BRCT beads (1 m g) and the specific radiolabeled BACH1 fragment. After extensive washing, the products retained on the beads were analyzed on SDS-polyacrylamide gels, followed by exposure to film. Each BACH1 fragment was generated as described in Materials and Methods. Those BACH1 fragments that specifically interacted with GST-BRCT are indicated by a "+" in the column to the right. (B) Further analysis of BACH1 residues 961-1008 revealed that it contained a smaller segment required for interaction with BRCA1 in vivo. Deletion of these 28 BACH1 residues (amino acids 979-1006), denoted by the Del28 species, resulted in loss of BRCA1 binding in vivo. Anti-myc (9E10) immunoprecipitations from 293 cells transfected with myc-tagged BACH1 full-length (FL), helicase domain (HD), and BACH1 lacking residues 979-1006 (Del28) (5-10 m g DNA each) were resolved on SDS-polyacrylamide gels and analyzed by Western blot for BRCA1 and BACH1 with anti-BRCA1 (MS110) and anti-BACH1 (E67) antibodies. (C) Schematic of full-length (FL) BACH1 depicting the 28-aa residues required for BRCA1 binding.