Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system

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.

MRN-dependent and independent pathways for recruitment of TOPBP1 to DNA double-strand breaks

“…ATR signaling requires one of two known activators, either TOPBP1 or ETAA1 [ 7 ]. Recent work from our group has shown that, in Xenopus egg extracts (XEEs), TOPBP1 is solely responsible for ATR activation at DSBs [ 8 , 9 ]. TOPBP1 is a multi-functional protein with roles in ATR signaling, the initiation of DNA replication, transcriptional regulation, and DNA repair during mitosis [ 10 ].…”

“…To study this important problem we utilized our recently developed DMAX system, which is based on XEEs and the use of linear dsDNA templates [ 8 , 9 ]. For DMAX, we use the high-speed supernatant (HSS) form of XEE, where traditionally prepared crude extracts are centrifuged at high speed to produce an extract containing just soluble protein, and devoid of pigment granules, yolk platelets, and membranes.…”

“…For DMAX, we use the high-speed supernatant (HSS) form of XEE, where traditionally prepared crude extracts are centrifuged at high speed to produce an extract containing just soluble protein, and devoid of pigment granules, yolk platelets, and membranes. To study ATR activation we simply add phage lambda DNA that had been digested with EcoRI (to produce six fragments, sizes ranging from 3.5 kb to 21 kb) to HSS, and this results in robust activation of both ATM and ATR [ 8 ]. The system is also amendable to studying how proteins associate with DSBs.…”

“…For this, we produce magnetic streptavidin beads that are linked to 5kb dsDNA molecules by virtue of a biotin group on one end of the dsDNAs. The 5kb size was chosen because linear dsDNAs that are any smaller fail to activate ATR in our system [ 8 ]. These “DSB beads” thus contain one free DNA end, which robustly activates both ATM and ATR [ 8 , 9 ].…”

“…The 5kb size was chosen because linear dsDNAs that are any smaller fail to activate ATR in our system [ 8 ]. These “DSB beads” thus contain one free DNA end, which robustly activates both ATM and ATR [ 8 , 9 ]. Beads are added to HSS, incubation ensues, and then the beads are recovered back out of the extract, washed, and probed for occupancy of proteins of interest.…”