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

The BRCA1 C-terminal (BRCT) domain is an interaction module that allows the host protein to bind to various biological substrates such as other proteins, nucleic acids, and poly (ADP-ribose) chains (reviewed by [8], [13], [23], [29]). BRCT proteins are major players in the DNA damage response (DDR), and it is therefore crucial that we understand how they function and that we fully describe all the different modes by which they interact with their binding partners. Regarding how BRCT domains bind to other proteins, there are two general categories: interactions that require the BRCT binding substrate to be phosphorylated, and those that do not. For phospho-driven interactions, some BRCT domains contain a so-called phosphate-binding pocket (PBP), which features a triad of conserved amino acid residues that interact with the phosphate group on the substrate protein (reviewed by [13], [23], [29], [4]). BRCT domains that bind phospho-epitopes are typically found in pairs, where one domain within the pair contains the PBP. While the PBP directly interacts with phosphate, both BRCT domains within the pair are required for binding as the PBP forms via intramolecular interactions between the two domains (reviewed by [17,29,8]). Intramolecular interactions between BRCT domains also allow binding of the host protein to substrate in a phospho-independent manner, and both the phospho-dependent and independent modes of binding have been observed within the same protein. The yeast Rtt107 protein contains 6 BRCT domains, with the C-terminal two BRCTs supplying phospho-dependent binding to histone H2A, and the four N-terminal domains forming a compact, higher-order assembly that binds peptide ligands from a number of proteins [30]. The net result is that Rtt107 binds to chromatin via the phospho-dependent mechanism, and then recruits other proteins to chromatin via the phospho-independent mechanism.

BRCT domains also make intermolecular contacts to allow assembly of both homo- and heterotypic multimers. A well-studied example of BRCT::BRCT intermolecular interaction is found with the XRCC1 and DNA Ligase 3 (LIG3) proteins [[1], [3], [6], [9], [14], [15], [27]]. Both proteins can homodimerize via BRCT::BRCT interaction, and the two can also heterodimerize via their BRCT domains. Recent work from our group was focused on the Topoisomerase II Binding Protein 1 (TOPBP1) protein, which contains nine BRCT domains, and we found that TOPBP1 forms oligomers and that the protein is capable of a series of BRCT::BRCT interactions [11]. More specifically, we found that BRCT1&2 could bind to BRCT domains 1,2, and 5, and that BRCT4&5 could bind BRCTs 1 and 2 but not 5. In this work we examine how TOPBP1 uses its BRCT domains to bind a heterologous protein, by studying its interaction with the NBS1 protein.

NBS1 is part of a trimeric complex, termed MRN for MRE11-RAD50-NBS1, that senses DNA double-strand breaks (DSBs) and then plays multiple roles in ensuring efficient repair of the breaks (reviewed by [16], [22], [26]). These roles include holding the two broken ends together, initiating DNA end resection, and activation of the DNA damage checkpoint kinases ATM and ATR. MRN activates ATM directly (reviewed by [20]), and ATR indirectly through its ability to recruit TOPBP1 to the DSB [18]. Recent work from our laboratory has shown that loss of MRN attenuates ATR signaling at DSBs, and TOPBP1′s occupancy at the DSB is also reduced [18]. TOPBP1 has a well-studied function in directly interacting with ATR (and its binding partner ATRIP) at sites of damage to stimulate ATR kinase activity (reviewed by [38]), and thus our recent work has revealed a pathway for ATR signaling that runs from the DNA end through MRN to TOPBP1 and then ATR [18]. One open and important question for this pathway is: what is the molecular mechanism by which MRN recruits TOPBP1 to DSBs?

In this study we focused on the NBS1 subunit as previous works have shown that TOPBP1 and NBS1 bind one another, and that this interaction is conserved from frogs to man [19], [33]. Interestingly, in both frogs and humans, TOPBP1 and NBS1 constitutively bind to one another, and the interaction is then enhanced during the DSB response [19], [33]. Here, we study the constitutive interaction, and we find that NBS1 uses its BRCT1 domain to make direct contacts with TOPBP1′s BRCT1 domain and, separately, the BRCT2 domain. Our data provide a molecular explanation for how MRN recruits TOPBP1 to DSBs, and they also expand our understanding of how BRCT::BRCT interactions drive the cellular response to DNA damage.

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