Huynh, O. | Search bioRxiv

bioRxiv2026

An NHEJ-independent Role for DNA-PKcs in ATR activation at DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) are threats to genome integrity, and to mitigate this risk cells activate the Ataxia Telangiectasia and Rad3-related (ATR) kinase, which halts cell cycle progression to allow time for repair. While ATR signalling during replication stress is well understood, how ATR is activated at DSBs remain unclear. Topoisomerase 2-Binding Protein 1 (TOPBP1) is a key activator of ATR, and activation is mediated by phosphorylation of TOPBP1 at Serine 1131 (S1131). Previous work showed that the Ataxia Telangiectasia Mutated (ATM) kinase phosphorylates TOPBP1 at S1131. ATM is primarily linked to the homologous recombination (HR)-based repair of DSBs, however the majority of cellular DSBs are repaired via the Non-Homologous End-Joining (NHEJ) repair pathway, raising the question of how (or if) ATR is activated in an ATM-independent manner. Here, using Xenopus egg extracts, we demonstrate that DNA-PKcs controls a pathway acting in parallel to ATM that promotes ATR signalling at DSBs. We show that, like ATM, DNA-PKcs phosphorylates TOPBP1 at S1131. DNA-PKcs is best known for orchestrating NHEJ, however we find that its roles in NHEJ and ATR signalling are separable. Our findings reveal an alternative pathway for ATR activation in which DNA-PKcs directly couples DSB recognition to ATR signalling. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/733426v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@4eb847org.highwire.dtl.DTLVardef@244193org.highwire.dtl.DTLVardef@4d4f4forg.highwire.dtl.DTLVardef@191de27_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell Biology↗

bioRxiv2022

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

The TOPBP1 and NBS1 proteins are key components of DNA repair and DNA-based signaling systems. TOPBP1 is a multi-BRCT domain containing protein that plays important roles in checkpoint signaling, DNA replication, and DNA repair. Likewise, NBS1, which is a component of the MRE11-RAD50-NBS1 (MRN) complex, functions in both checkpoint signaling and DNA repair. NBS1 also contains BRCT domains, and previous works have shown that TOPBP1 and NBS1 interact with one another. In this work we examine the interaction between TOPBP1 and NBS1 in detail. We report that NBS1 uses its BRCT1 domain to interact with TOPBP1s BRCT1 domain and, separately, with TOPBP1s BRCT2 domain. Thus, NBS1 can make two distinct contacts with TOPBP1. We report that recombinant TOPBP1 and NBS1 proteins bind one another in a purified system, showing that the interaction is direct and does not require post-translational modifications. Surprisingly, we also report that intact BRCT domains are not required for these interactions, as truncated versions of the domains are sufficient to confer binding. For TOPBP1, we find that small 24-29 amino acid sequences within BRCT1 or BRCT2 allow binding to NBS1, in a transferrable manner. These data expand our knowledge of how the crucial DNA damage response proteins TOPBP1 and NBS1 interact with one another and set the stage for functional analysis of the two disparate binding sites for NBS1 on TOPBP1.

cell biology↗