Structure of a Second BRCT Domain Identified in the Nijmegen Breakage Syndrome Protein Nbs1 and its Function in an MDC1-Dependent Localization of Nbs1 to DNA Damage Sites

Abstract

The Nijmegen breakage syndrome protein Nbs1 is a component of the MRN (Mre11-Rad50-Nbs1) complex, central to the DNA damage response. While Nbs1 is generally believed to encompass a forkhead-associated domain linked to a breast cancer C-terminal (BRCT) domain, to date there is no experimental information on its three-dimensional structure. Through nuclear magnetic resonance (NMR) three-dimensional structure determination, we demonstrate that there is a second BRCT domain (BRCT2) in Nbs1. The domain has the characteristic BRCT topology, but with a long insertion shown to be flexible by NMR relaxation measurements. In the absence of sequence similarity to other proteins, a search for structural analogs of BRCT2 returned the second BRCT domain of the tandem BRCT repeats of cell cycle checkpoint proteins MDC1 (mediator of DNA damage checkpoint protein 1) and BRCA1 (breast cancer protein 1), suggesting that like MDC1 and BRCA1, Nbs1 also possesses tandem BRCT domains with phosphoprotein binding ability. Structurebased single point mutations in human Nbs1 were evaluated in vivo and revealed that BRCT2 is essential for an MDC1-dependent relocalization of Nbs1 to DNA damage sites, most likely through a direct interaction of Nbs1 tandem BRCT domains with phosphorylated MDC1.

Molecular Basis for the Association of Microcephalin (MCPH1) Protein with the Cell Division Cycle Protein 27 (Cdc27) Subunit of the Anaphase-promoting Complex

“…In vitro phosphopeptide interaction screens along with the crystal structures of BRCA1-and MDC1-phosphopeptide complexes have helped define a consensus BRCT binding phosphoprotein interaction site (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). This consensus binding site conforms to the pS/XXX rule where pS is a phosphoserine and X is any residue, with position pSϩ3 usually being a phenylalanine (BRCA1) or tyrosine (MDC1) or an aliphatic residue (TopBP1, Brc1, and Crb2), and possibly an aspartate in the sequence recognized by NBS1 tandem BRCT domains (24). In the case of TopBP1 the C-terminal tandem BRCT domains can bind either phosphoserine-or phosphothreonine-containing peptides (23).…”

MDC1: The art of keeping things in focus

“…Mutation of key residues in the FHA and the tandem BRCT domain similarly reduce the interaction between CK2-phosphorylated MDC1 protein and the MRN complex, thus indicating the requirement of an intact domain structure at the N-terminus of NBS1 for optimal binding (Chapman and Jackson 2008;Lloyd et al 2009). Moreover, alterations in either FHA or BRCT domains impair relocalization of the MRN complex to IRIF (Cerosaletti and Concannon 2003;Spycher et al 2008;Wu et al 2008;Xu et al 2008). These data point to a dual phospho-dependent interaction mode between MDC1 and the FHA/BRCT region of NBS1.…”

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

“…The FHA domain is a modular phosphopeptide recognition motif [402,403]. Subsequent studies confirm the importance of both the Nterminal FHA and tandem BRCT domains of NBS1 for its interaction with MDC1 and accumulation at sites of IR-induced DSBs [404][405][406][407]. The N-terminal region of human MDC1 containing six SDTD motifs, which correspond to consensus CK2 phosphorylation sites, is constitutively phosphorylated and mediates constitutive binding to the FHA and BRCT domains of NBS1 [404,405,[408][409][410].…”