BRCT domain interactions in the heterodimeric DNA repair protein XRCC1-DNA ligase III

Proteins involved in DNA repair, or its coordination with DNA replication and mitosis through cell cycle checkpoints, are vital in the concerted cellular response to DNA damage that maintains the integrity of the genome. The "BRCT" domain (BRCA1 carboxy terminal) was noted as a putative protein-protein interaction motif in the breast cancer suppressor gene, BRCA1, and subsequently identified in over 50 proteins involved in DNA repair, recombination, or cell cycle control. The heterodimer of the DNA repair proteins, XRCC1 and DNA ligase III, was the first example of a functional interaction via BRCT modules. The only three-dimensional crystal structure of a BRCT domain was solved for this region of XRCC1. Key amino acid residues mediating the interaction with DNA ligase III were identified here by targeted mutagenesis of the XRCC1 BRCT domain. The consequences of these mutations on protein folding were assessed. A structural model of the DNA ligase III BRCT domain was constructed and similarly tested by mutation of corresponding residues required for the interaction with XRCC1. These data identify the XRCC1-DNA ligase III heterodimer interface and provide the first demonstration of the surface contacts coordinating a functional BRCT-BRCT protein interaction.     

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     

Multimodal approach to explore the pathogenicity of BARD1, ARG 658 CYS,and ILE 738 VAL mutants

BARD1–BRCA1 complex plays an important role in DNA damage repair, apoptosis, chromatin remodeling, and other important processes required for cell survival. BRCA1 and BARD1 heterodimer possess E3 ligase activity and is involved in genome maintenance, by functioning in surveillance for DNA damage, thereby regulating multiple pathways including tumor suppression. BRCT domains are evolutionary conserved domains present in different proteins such as BRCA1, BARD1, XRCC, and MDC1 regulating damage response and cell-cycle control through protein–protein interactions. Nonetheless, the role of BARD1BRCT in the recruitment of DNA repair mechanism and structural integrity with BRCA1 complex is still implicit. To explicate the role of BARD1BRCT in the DNA repair mechanism, in silico, in vitro, and biophysical approach were applied to characterize BARD1 BRCT wild-type and Arg658Cys and Ile738Val mutants. However, no drastic secondary and tertiary structural changes in the mutant proteins were observed. Thermal and chemical denaturation studies revealed that mutants Arg658Cys and Ile738Val have a decrease in T_m and ?G than the wild type. In silico studies of BARD1 BRCT (568-777) and mutant protein indicate loss in structural compactness on the Ile738Val mutant. Comparative studies of wild-type and mutants will thus be helpful in understanding the basic role of BARD1BRCT in DNA damage repair.     

Cancer predisposing mutations in BRCT domains

Members of the breast cancer 1 (BRCA1) carboxy-terminal (BRCT) superfamily are involved in the cellular response to the DNA damage sensing and repair, as well as in the cell cycle control. All proteins are characterized by one or more BRCT domain(s), which provides a flexible framework representing scaffolding element(s) in multi-protein complexes. In particular, BRCA1, nibrin (NBN), and microcephalin (MCPH1), generally considered as molecular models for cancer-prone syndromes, contain BRCT domains able to bind phosphorylated proteins. Mutations within the BRCT domains of BRCA1, NBN, and MCPH1 are responsible for cancer susceptibility, both at the homozygous and heterozygous status. Here, we report a critical analysis of: (i) the BRCT domain structure, (ii) the role of BRCA1, NBN, and MCPH1 in DNA damage sensing and repair as well as in cell cycle control, and (iii) the pathological effects of mutations within the BRCT domains of BRCA1, NBN, and MCPH1.     

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.     

Structural basis for phosphorylation-dependent signaling in the DNA-damage response.

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.     

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals structural similarity to the BRCT domains of polymerase mu and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol lambda plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. In conclusion, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.     

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.     

The BRCA1 C-terminal domain: structure and function

The BRCA1 C-terminal region contains a duplicated globular domain termed BRCT that is found within many DNA damage repair and cell cycle checkpoint proteins. The unique diversity of this domain superfamily allows BRCT modules to interact forming homo/hetero BRCT multimers, BRCT-non-BRCT interactions, and interactions with DNA strand breaks. The sequence and functional diversity of the BRCT superfamily suggests that BRCT domains are evolutionarily convenient interaction modules.     

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.     

Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIalpha (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity.     

RAD18-BRCTx interaction is required for efficient repair of UV-induced DNA damage

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage signaling pathways. The BRCT domain-containing protein BRCTx has been shown to interact physically with RAD18, an E3 ligase involved in postreplication repair and homologous recombination repair. However, the physiological relevance of the interaction between RAD18 and BRCTx is largely unknown. In this study, we showed that RAD18 interacts with BRCTx in a phosphorylation-dependent manner and that this interaction, mediated via highly conserved serine residues on the RAD18 C terminus, is required for BRCTx accumulation at DNA damage sites. Furthermore, we uncovered critical roles of the RAD18-BRCTx module in UV-induced DNA damage repair but not PCNA mono-ubiquitination or homologous recombination. Thus, our results suggest that RAD18 has an additional function in the surveillance of the UV-induced DNA damage response signal.     

Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains

BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding.     

Conformational Effects of a Common Codon 399 Polymorphism on the BRCT1 Domain of the XRCC1 Protein

The X-ray cross-complementing-1 (XRCC1) protein functions as a scaffold that coordinates the activity of the cellular machinery involved in base excision repair (BER) of DNA damage. The BRCT1 domain of XRCC1 is responsible for interacting with several of the key components of the BER machinery, and it is also the site of a common genetic polymorphism in XRCC1 at amino acid residue 399 (Arg → Gln). Experimental and epidemiologic evidence suggest that this polymorphism may alter BER capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XRCC1 induced by the polymorphism. Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the BRCT1 domain of XRCC1, and differences in structure produced by the polymorphic substitution were determined. The results indicate that, although the general configuration of both proteins is similar and there is little actual deviation at the site of the polymorphism itself, the substitution produces significant conformational changes at several other sites in the BRCT1 domain, including the loss of secondary structural features such as α helices that may be critical for protein–protein interactions. These results provide support for the hypothesis that this polymorphism in XRCC1 could affect DNA repair capability by altering the structure of the BRCT1 domain and thus the ability of XRCC1 to coordinate BER.     

Interactions between BRCT repeats and phosphoproteins: tangled up in two

The C-terminal region of the breast-cancer-associated protein BRCA1 contains a pair of tandem BRCA1 C-terminal (BRCT) repeats that are essential for the tumour suppressor function of the protein. Similar repeat sequences have been identified in many proteins that seem to mediate cellular mechanisms for dealing with DNA damage. The BRCT domain in BRCA1 has been recently shown to constitute a module for recognizing phosphorylated (phospho-) peptides, with a recognition groove that spans both BRCT repeats. The fact that many other BRCT-containing proteins have phospho-peptide binding activity suggests that BRCT repeats might mediate phosphorylation-dependent protein-protein interactions in processes that are central to cell-cycle checkpoint and DNA repair functions.     

A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.     

Solution structure of polymerase mu's BRCT Domain reveals an element essential for its role in nonhomologous end joining

The solution structure and dynamics of the BRCT domain from human DNA polymerase mu, implicated in repair of chromosome breaks by nonhomologous end joining (NHEJ), has been determined using NMR methods. BRCT domains are typically involved in protein-protein interactions between factors required for the cellular response to DNA damage. The pol mu BRCT domain is atypical in that, unlike other reported BRCT structures, the pol mu BRCT is neither part of a tandem grouping, nor does it appear to form stable homodimers. Although the sequence of the pol mu BRCT domain has some unique characteristics, particularly the presence of >10% proline residues, it forms the characteristic alphabetaalpha sandwich, in which three alpha helices are arrayed around a central four-stranded beta-sheet. The structure of helix alpha1 is characterized by two solvent-exposed hydrophobic residues, F46 and L50, suggesting that this element may play a role in mediating interactions of pol mu with other proteins. Consistent with this argument, mutation of these residues, as well as the proximal, conserved residue R43, specifically blocked the ability of pol mu to efficiently work together with NHEJ factors Ku and XRCC4-ligase IV to join noncomplementary ends together in vitro. The structural, dynamic, and biochemical evidence reported here identifies a functional surface in the pol mu BRCT domain critical for promoting assembly and activity of the NHEJ machinery. Further, the similarity between the interaction regions of the BRCT domains of pol mu and TdT support the conclusion that they participate in NHEJ as alternate polymerases.