An atypical BRCT–BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.     

Structure of an XRCC1 BRCT domain: a new protein-protein interaction module.

The BRCT domain (BRCA1 C-terminus), first identified in the breast cancer suppressor protein BRCA1, is an evolutionarily conserved protein-protein interaction region of approximately 95 amino acids found in a large number of proteins involved in DNA repair, recombination and cell cycle control. Here we describe the first three-dimensional structure and fold of a BRCT domain determined by X-ray crystallography at 3.2 A resolution. The structure has been obtained from the C-terminal region of the human DNA repair protein XRCC1, and comprises a four-stranded parallel beta-sheet surrounded by three alpha-helices, which form an autonomously folded domain. The compact XRCC1 structure explains the observed sequence homology between different BRCT motifs and provides a framework for modelling other BRCT domains. Furthermore, the established structure of an XRCC1 BRCT homodimer suggests potential protein-protein interaction sites for the complementary BRCT domain in DNA ligase III, since these two domains form a stable heterodimeric complex. Based on the XRCC1 BRCT structure, we have constructed a model for the C-terminal BRCT domain of BRCA1, which frequently is mutated in familial breast and ovarian cancer. The model allows insights into the effects of such mutations on the fold of the BRCT domain.     

Receptor-like protein tyrosine phosphatase alpha homodimerizes on the cell surface

We reported previously that the N-terminal D1 catalytic domain of receptor protein-tyrosine phosphatase alpha (RPTPalpha) forms a symmetrical, inhibited dimer in a crystal structure, in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer. Previous functional studies also suggested that dimerization inhibits the biological activity of a CD45 chimeric RPTP and the catalytic activity of an isolated RPTPsigma D1 catalytic domain. Most recently, we have also shown that enforced dimerization inhibits the biological activity of full-length RPTPalpha in a wedge-dependent manner. The physiological significance of such inhibition is unknown, due to a lack of understanding of how RPTPalpha dimerization is regulated in vivo. In this study, we show that transiently expressed cell surface RPTPalpha exists predominantly as homodimers, suggesting that dimerization-mediated inhibition of RPTPalpha biological activity is likely to be physiologically relevant. Consistent with our published and unpublished crystallographic data, we show that mutations in the wedge region of D1 catalytic domain and deletion of the entire D2 catalytic domain independently reduced but did not abolish RPTPalpha homodimerization, suggesting that both domains are critically involved but that neither is essential for homodimerization. Finally, we also provide evidence that both the RPTPalpha extracellular domain and the transmembrane domain were independently able to homodimerize. These results lead us to propose a zipper model in which inactive RPTPalpha dimers are stabilized by multiple, relatively weak dimerization interfaces. Dimerization in this manner would provide a potential mechanism for negative regulation of RPTPalpha. Such RPTPalpha dimers could be activated by extracellular ligands or intracellular binding proteins that induce monomerization or by intracellular signaling events that induce an open conformation of the dimer.     

Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains

Protein phosphorylation by protein kinases may generate docking sites for other proteins. It thus allows the assembly of signaling complexes in response to kinase activation. Several protein domains that bind phosphoserine or phosphothreonine residues have been identified, including the 14-3-3, PIN1, FHA, KIX, WD-40 domain, and polo box (Yaffe, M. B., and Elia, A. E. (2001) Curr. Opin. Cell Biol. 13, 131-138; Elia, A. E., Cantley, L. C., and Yaffe, M. B. (2003) Science 299, 1228-1231). The BRCA1 COOH-terminal (BRCT) domains are protein modules found in many proteins that regulate DNA damage responses (Koonin, E. V., Altschul, S. F., and Bork, P. (1996) Nat. Genet. 13, 266-268). Whether BRCT domains can mediate phosphorylation-dependent interactions has not been systematically investigated. We report here that the BRCT domains also recognize phosphopeptides. Oriented peptide library analysis indicated that the BRCT domains from BRCA1, MDC1, BARD1, and DNA Ligase IV preferred distinct phosphoserine-containing peptides. In addition, the interaction between BRCA1 and the BRCT binding motif of BACH1 was required for BACH1 checkpoint activity. Furthermore, BRCT domains of the yeast DNA repair protein Rad9 could bind phosphopeptides, suggesting that the BRCT domains represent a class of ancient phosphopeptide-binding modules. Potential targets of BRCT domains were identified through data base search. Structural analysis of BRCA1 BRCT repeats also predicted conserved residues that may form the phosphopeptide-binding pocket. Thus, the BRCT repeats are a new family of phosphopeptide-binding domains in DNA damage responses.     

Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein

Protein interactions critical to DNA repair and cell cycle control systems are often coordinated by modules that belong to a superfamily of structurally conserved BRCT domains. Because the mechanisms of BRCT interactions and their significance are not well understood, we sought to define the affinity and specificity of those BRCT modules that orchestrate base excision repair and single-strand break repair. Common to these pathways is the essential XRCC1 DNA repair protein, which interacts with at least nine other proteins and DNA. Here, we characterized the interactions of four purified BRCT domains, two from XRCC1 and their two partners from DNA ligase IIIalpha and poly(ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase IIIalpha BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase IIIalpha BRCT complex. To examine the assembly states of isolated BRCT domains and pairwise domain complexes, we used size-exclusion chromatography coupled with on-line light scattering. This analysis indicated that isolated BRCT domains form homo-oligomers and that the BRCT complex between the C-terminal XRCC1 domain and the ligase IIIalpha domain is a heterotetramer with 2:2 stoichiometry. Using affinity capture and surface plasmon resonance methods, we determined that specific heteromeric interactions with high nanomolar dissociation constants occur between pairs of cognate BRCT domains. A structural model for a XRCC1 x DNA ligase IIIalpha heterotetramer is proposed as a core base excision repair complex, which constitutes a scaffold for higher order complexes to which other repair proteins and DNA are brought into proximity.     

Identification of the Interactors of Human Nibrin (NBN) and of Its 26 kDa and 70 kDa Fragments Arising from the NBN 657del5 Founder Mutation

Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD⁺ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at 24 h from IR. Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS.     

BRCT domains: Easy as one,two, three

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero-domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.Key words: BRCT domain, DNA repair, phosphorylation, phospho-peptide interaction, protein interaction, DNA binding, DNA damage response     

Rb-associated protein 46 (RbAp46) inhibits transcriptional transactivation mediated by BRCA1

The retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46) is a member of the WD-repeat protein family and a component of the histone modifying and remodeling complexes. Previously, we demonstrated that RbAp46 is a potent growth inhibitor that can suppress the transformed phenotype of tumor cells. To explore the molecular mechanisms of RbAp46 function, we used RbAp46 as a bait in a yeast two-hybrid screening and found that RbAp46 interacts specifically with the C-terminal region of BRCA1 (the BRCT domain), a domain involved in the t transactivation activity of BRCA1. Coimmunoprecipitation assays demonstrated that the interaction of RbAp46 with BRCA1 requires the first two of the four Trp-Asp (WD)-repeats of RbAp46. We also showed that expression of RbAp46 represses the transactivation activity mediated by the BRCT/Gal4 fusion protein and inhibits the transactivation of the p21 promoter mediated by the full-length BRCA1. Interestingly, the association of BRCA1 and RbAp46 is disrupted in cells treated with DNA-damaging agents. These results suggest that RbAp46 may specifically interact with BRCA1 and modulate its transactivation activity in response to DNA damage.     

Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra α-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5′-phosphate recognition by the BRCT domains of bacterial NAD⁺-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions.     

Characterization of the DNA binding and structural properties of the BRCT region of human replication factor C p140 subunit

BRCT domains, present in a large number of proteins that are involved in cell cycle regulation and/or DNA replication or repair, are primarily thought to be involved in protein-protein interactions. The large (p140) subunit of replication factor C contains a sequence of approximately 100 amino acids in the N-terminal region that binds DNA and is distantly related to known BRCT domains. Here we show that residues 375-480, which include 28 amino acids N-terminal to the BRCT domain, are required for 5'-phosphorylated double-stranded DNA binding. NMR chemical shift analysis indicated that the N-terminal extension includes an alpha-helix and confirmed the presence of a conserved BRCT domain. Sequence alignment of the BRCT region in the p140 subunit of replication factor C from various eukaryotes has identified very few absolutely conserved amino acid residues within the core BRCT domain, whereas none were found in sequences immediately N-terminal to the BRCT domain. However, mapping of the limited number of conserved, surface-exposed residues that were found onto a homology model of the BRCT domain, revealed a clustering on one side of the molecular surface. The cluster, as well as a number of amino acids in the N-terminal alpha-helix, were mutagenized to determine the importance for DNA binding. To ensure minimal structural changes because of the introduced mutations, proteins were checked using one-dimensional (1)H NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal alpha-helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.     

Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA)

NAD(+)-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD(+)-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.     

BRCT domains

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero- domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.     

Solution structure of Rap1 BRCT domain from Saccharomyces cerevisiae reveals a novel fold

Rap1 (repressor-activator protein 1) from Saccharomyces cerevisiae, containing a BRCT domain at its N-terminus, is a multifunctional protein that controls telomere function, silencing, and the activation of glycolytic and ribosomal protein genes. In this work, we determined the solution structure of Rap1 BRCT domain, which contains three β-strands and three α-helices. Structural comparison indicated that Rap1 BRCT domain adopts a global fold similar to other BRCT domains, implying some common structural aspects of BRCT domain family. On the other hand, Rap1 BRCT domain displays structural characteristics significantly different from other BRCT domains in that Rap1 BRCT domain adopts a rather flexible conformation with less secondary structure elements, revealing a novel fold of the BRCT domain family.     

BRCA1-directed,enhanced and aberrant homologous recombination: Mechanism and potential treatment strategies

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.Key words: BRCT, DNA repair, peptide, radiation, RING, ubiquitylation