Abstract
The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1's nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.