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

Abstract

Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase is activated by DNA replication stress and also by various forms of DNA damage, including DNA double-strand breaks (DSBs). Recruitment to sites of damage is insufficient for ATR activation as one of two known ATR activators, either topoisomerase II-binding protein (TOPBP1) or Ewing’s tumor-associated antigen 1, must also be present for signaling to initiate. Here, we employ our recently established DSB-mediated ATR activation in Xenopus egg extract (DMAX) system to examine how TOPBP1 is recruited to DSBs, so that it may activate ATR. We report that TOPBP1 is only transiently present at DSBs, with a half-life of less than 10 minutes. We also examined the relationship between TOPBP1 and the MRE11-RAD50-NBS1 (MRN), CtBP interacting protein (CtIP), and Ataxia Telangiectasia mutated (ATM) network of proteins. Loss of MRN prevents CtIP recruitment to DSBs, and partially inhibits TOPBP1 recruitment. Loss of CtIP has no impact on either MRN or TOPBP1 recruitment. Loss of ATM kinase activity prevents CtIP recruitment and enhances MRN and TOPBP1 recruitment. These findings demonstrate that there are MRN-dependent and independent pathways that recruit TOPBP1 to DSBs for ATR activation. Lastly, we find that both the 9-1-1 complex and MDC1 are dispensable for TOPBP1 recruitment to DSBs.

NBS1 binds directly to TOPBP1 via disparate interactions between the NBS1 BRCT1 domain and the TOPBP1 BRCT1 and BRCT2 domains

“…Finally, Yoo and colleagues showed that mutations in the PBPs from both TOPBP1 (BRCT1) and NBS1 (BRCT1) prevented efficient interaction. We have recently shown that MRN is important for recruitment of TOPBP1 to long, linearized dsDNA templates that mimic DSBs (Montales et al, 2022). Given these findings, we were motivated to analyze the TOPBP1-NBS1 interaction in more detail.…”

“…Accordingly, we expressed and purified from E. coli two GST-NBS1 1-400 fusion proteins, one corresponding to the wild type sequence and the other containing a K158E mutation that attenuates the PBP in BRCT1 of NBS1 (Figure 1A). These proteins, along with GST alone, were added to XEE in either the presence or absence of linearized 5kb dsDNA, which we have previously shown will potently activate both ATR and ATM in our XEEs (Montales et al, 2021; Ruis et al, 2022; Montales et al, 2022). The GST protein complexes were then recovered back out of the XEE using glutathione-sepharose beads, and the presence of endogenous TOPBP1 was detected via Western blotting.…”

“…In this work we extend our understanding of BRCT::BRCT interactions by studying how TOPBP1 interacts with the NBS1 protein. We chose this interaction to study because recent work from our group showed that NBS1, a component of the critical DNA repair factor MRN, plays an important role in recruiting TOPBP1 to sites of damage (Montales et al, 2022). Here, we report that the NBS1::TOPBP1 interaction is driven by BRCT::BRCT interactions, whereby the BRCT1 domain from NBS1 binds directly to the both the BRCT1 and BRCT2 domains of TOPBP1.…”

Multi-site phosphorylation of the TOPBP1 ATR Activation Domain propels ATR signaling during the response to DNA breaks

“…The beads are then incubated in XEE containing myc-tagged forms of either wild type or S1131A TOPBP1. Since our previous work has repeatedly shown that TOPBP1 does not bind to streptavidin beads lacking DNA under these assay conditions (Montales et al, 2021; Ruis et al, 2022; Montales et al, 2022), we did not include empty beads in this experiment. After incubation, the beads were isolated, washed, and probed for TOPBP1 occupancy by Western blotting.…”

“…In this work we use our D SB- m ediated A TR activation in X enopus egg extract system (DMAX; Montales et al, 2021; Ruis et al, 2022; Montales et al, 2022) to study how TOPBP1’s AAD is regulated. DMAX offers several powerful advantages for mechanistic studies of ATR signaling.…”