Delineation of a minimal topoisomerase II binding protein 1 for regulated activation of ATR at DNA double-strand breaks

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

“…Our previous work had shown that addition of a recombinant protein corresponding to the TOPBP1 BRCT0-2 region to XEE, at 1 uM (approximately 25-fold excess over endogenous TOPBP1), efficiently blocks ATR activation [ 8 ]. Furthermore, other work from our group has shown that sole function of the BRCT0-2 region is to recruit TOPBP1 to DSBs [ 9 ], and work from others has shown that human TOPBP1 uses it BRCT0-2 region to gain entry into damage-induced, sub-nuclear foci in G1 cells [ 13 ]. It thus stands to reason that excess recombinant BRCT0-2 blocks ATR activation by preventing recruitment of endogenous TOPBP1 to DSBs.…”

“…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 ].…”

“…It is comprised of nine copies of the BRCA1 C-Terminal (BRCT) domain, as well as an ATR activation domain (AAD), located between BRCT6 and BRCT7 ( Fig 1 ). Recent work from our group has delineated a minimal, synthetic form of TOPBP1, named Junior, that is competent for ATR activation by DSBs [ 9 ]. To create Junior the sequences spanning BRCT3 to BRCT6 were removed, and it was shown that the remaining sequences are sufficient for regulated activation of ATR ( Fig 1 ).…”

“…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.…”

“…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.…”