BRCA1 C-terminal Research Articles

Loss of Rereplication Control inSaccharomyces cerevisiaeResults in Extensive DNA Damage

To maintain genome stability, the entire genome of a eukaryotic cell must be replicated once and only once per cell cycle. In many organisms, multiple overlapping mechanisms block rereplication, but the consequences of deregulating these mechanisms are poorly understood. Here, we show that disrupting these controls in the budding yeast Saccharomyces cerevisiae rapidly blocks cell proliferation. Rereplicating cells activate the classical DNA damage-induced checkpoint response, which depends on the BRCA1 C-terminus checkpoint protein Rad9. In contrast, Mrc1, a checkpoint protein required for recognition of replication stress, does not play a role in the response to rereplication. Strikingly, rereplicating cells accumulate subchromosomal DNA breakage products. These rapid and severe consequences suggest that even limited and sporadic rereplication could threaten the genome with significant damage. Hence, even subtle disruptions in the cell cycle regulation of DNA replication may predispose cells to the genomic instability associated with tumorigenesis.

Effects of Deletion and Site-Directed Mutations on Ligation Steps of NAD+-Dependent DNA Ligase: A Biochemical Analysis of BRCA1 C-Terminal Domain

DNA strand joining entails three consecutive steps: enzyme adenylation to form AMP-ligase, substrate adenylation to form AMP-DNA, and nick closure. In this study, we investigate the effects on ligation steps by deletion and site-directed mutagenesis of the BRCA1 C-terminal (BRCT) domain using NAD(+)-dependent DNA ligase from Thermus species AK16D. Deletion of the BRCT domain resulted in substantial loss of ligation activity, but the mutant was still able to form an AMP-ligase intermediate, suggesting that the defects caused by deletion of the entire BRCT domain occur primarily at steps after enzyme adenylation. The lack of AMP-DNA accumulation by the domain deletion mutant as compared to the wild-type ligase indicates that the BRCT domain plays a role in the substrate adenylation step. Gel mobility shift analysis suggests that the BRCT domain and helix-hairpin-helix subdomain play a role in DNA binding. Similar to the BRCT domain deletion mutant, the G617I mutant showed a low ligation activity and lack of accumulation of AMP-DNA intermediate. However, the G617I mutant was only weakly adenylated, suggesting that a point mutation in the BRCT domain could also affect the enzyme adenylation step. The significant reduction of ligation activity by G634I appears to be attributable to a defect at the substrate adenylation step. The greater ligation of mismatched substrates by G638I is accountable by accelerated conversion of the AMP-DNA intermediate to a ligation product at the final nick closure step. The mutational effects of the BRCT domain on ligation steps in relation to protein-DNA and potential protein-protein interactions are discussed.

Homo-oligomerization Is the Essential Function of the Tandem BRCT Domains in the Checkpoint Protein Crb2

BRCT (BRCA1 C terminus) domains are frequently found as a tandem repeat in proteins involved in DNA damage responses, such as Saccharomyces cerevisiae Rad9, human 53BP1 and BRCA1. Tandem BRCT domains mediate protein-protein and protein-DNA interactions. However, the functional significance of these interactions is largely unknown. Here we report the oligomerization of Schizosaccharomyces pombe checkpoint protein Crb2 through its tandem BRCT domains. Truncated Crb2 without BRCT domains is defective in DNA damage checkpoint signaling. However, addition of either of two heterologous dimerization motifs largely restores the functions of truncated Crb2 without BRCT domains. Replacement of Crb2 BRCT domains with a dimerization motif also renders cells resistant to the dominant negative effect of overexpressing Crb2 BRCT domains. These results demonstrate that the crucial function of the tandem BRCT domains is to oligomerize Crb2.

Poly(ADP-ribose) polymerase 1 regulates both the exonuclease and helicase activities of the Werner syndrome protein.

Werner syndrome (WS) is a genetic premature aging disorder in which patients appear much older than their chronological age. The gene mutated in WS encodes a nuclear protein (WRN) which possesses 3'-5' exonuclease and ATPase-dependent 3'-5' helicase activities. The genomic instability associated with WS cells and the biochemical characteristics of WRN suggest that WRN plays a role in DNA metabolic pathways such as transcription, replication, recombination and repair. Recently we have identified poly(ADP-ribose) polymerase-1 (PARP-1) as a new WRN interacting protein. In this paper, we further mapped the interacting domains. We found that PARP-1 bound to the N-terminus of WRN and to the C-terminus containing the RecQ-conserved (RQC) domain. WRN bound to the N-terminus of PARP-1 containing DNA binding and BRCA1 C-terminal (BRCT) domains. We show that unmodified PARP-1 inhibited both WRN exonuclease and helicase activities, and to our knowledge is the only known WRN protein partner that inactivates both of the WRN's catalytic activities suggesting a biologically significant regulation. Moreover, this dual inhibition seems to be specific for PARP-1, as PARP-2 did not affect WRN helicase activity and only slightly inhibited WRN exonuclease activity. The differential effect of PARP-1 and PARP-2 on WRN catalytic activity was not due to differences in affinity for WRN or the DNA substrate. Finally, we demonstrate that the inhibition of WRN by PARP-1 was influenced by the poly(ADP-ribosyl)ation state of PARP-1. The biological relevance of the specific modulation of WRN catalytic activities by PARP-1 are discussed in the context of pathways in which these proteins may function together, namely in the repair of DNA strand breaks.

Structure-based assessment of missense mutations in human BRCA1: implications for breast and ovarian cancer predisposition.

The BRCA1 gene from individuals at risk of breast and ovarian cancers can be screened for the presence of mutations. However, the cancer association of most alleles carrying missense mutations is unknown, thus creating significant problems for genetic counseling. To increase our ability to identify cancer-associated mutations in BRCA1, we set out to use the principles of protein three-dimensional structure as well as the correlation between the cancer-associated mutations and those that abolish transcriptional activation. Thirty-one of 37 missense mutations of known impact on the transcriptional activation function of BRCA1 are readily rationalized in structural terms. Loss-of-function mutations involve nonconservative changes in the core of the BRCA1 C-terminus (BRCT) fold or are localized in a groove that presumably forms a binding site involved in the transcriptional activation by BRCA1; mutations that do not abolish transcriptional activation are either conservative changes in the core or are on the surface outside of the putative binding site. Next, structure-based rules for predicting functional consequences of a given missense mutation were applied to 57 germ-line BRCA1 variants of unknown cancer association. Such a structure-based approach may be helpful in an integrated effort to identify mutations that predispose individuals to cancer.

Structure of the BRCT Repeats of BRCA1 Bound to a BACH1 Phosphopeptide: Implications for Signaling

The recognition of the phosphorylated BACH1 helicase by the BRCA1 C-terminal (BRCT) repeats is important to the tumor suppressor function of BRCA1. Here we report the crystal structure of the BRCT repeats of human BRCA1 bound to a phosphorylated BACH1 peptide at 2.3 Å resolution. The phosphorylated serine 990 and phenylalanine 993 of BACH1 anchor the binding to BRCA1 through specific interactions with a surface cleft at the junction of the two BRCT repeats. This surface cleft is highly conserved in BRCA1 across species, suggesting an evolutionarily conserved function of phosphopeptide recognition. Importantly, conserved amino acids critical for BACH1 binding are frequently targeted for missense mutations in breast cancer. These mutations greatly diminish the ability of BRCA1 to interact with the phosphorylated BACH1 peptide. Additional structural analysis revealed significant implications for understanding the function of the BRCT family of proteins in DNA damage and repair signaling.

Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination.

The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.

Mapping BRCT Interactions

The BRCT (BRCA1 C-terminal) domain is an amino acid motif found in BRCA1, a tumor suppressor protein (mutations of which are associated with breast and ovarian cancer). This motif is also found in many other proteins implicated in regulating DNA damage and checkpoint responses. Recent research has identified the BRCT domain as a phosphoserine/phosphothreonine-dependent binding motif that may recruit proteins to sites phosphorylated by kinases activated in response to DNA damage [(see Editorial Guide and Sci. STKE , 2003 , tw421 (2003)]. Rodriguez et al. used oriented phosphoserine-containing peptide libraries to analyze the binding specificity of the BRCT domains of BRCA1, MDC1 (a protein involved in DNA damage responses), BARD1 (whose mutation is associated with breast cancer), and DNA ligase IV. Fusion proteins containing tandem BRCT repeats from these proteins preferentially bound to distinct phosphoserine-containing peptides. The tandem BRCT domains of Rad9, a DNA repair protein from yeast, also bound phosphopeptides, which indicated that this interaction had arisen early in evolution. Mutational analysis of the BRCA1 BRCT-binding motif in the target BACH1 (BRCA1-associated carboxyl-terminal helicase 1) transfected into HeLa cells indicated that this motif was important for the G 2 /M checkpoint response to DNA damage. Database searches based on the consensus motifs identified potential targets of the various BRCT domain-containing proteins. These data further implicate the BRCT domain-containing proteins in the establishment of phosphorylation-dependent protein complexes involved in the DNA damage response. M. Rodriguez, X. Yu, J. Chen, Z. Songyang, Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains. J. Biol. Chem . 278 , 52914-52918 (2003). [Abstract] [Full Text]

The telomeric protein Rap1 is conserved in vertebrates and is expressed from a bidirectional promoter positioned between the Rap1 and KARS genes

We have identified the chicken homolog of the mammalian telomere protein repression and activation protein 1 (Rap1). Although cRap1 has only 36% sequence identity to hRap1, it contains the same conserved BRCA1 C- terminal (BRCT), Myb and Rap C- terminus (RCT) domains. Two-hybrid analysis and immunolocalization experiments revealed that cRap1 interacts with the telomere-binding protein telomeric repeat binding factor (TRF)2 and localizes to telomeres. Thus, despite considerable sequence divergence, the identity and overall domain structure of telomere-associated proteins is conserved in vertebrates. Analysis of the cRap1 genomic locus revealed that the cRap1 gene lies immediately adjacent to the cKARS (lysyl-tRNA synthetase) gene with the two genes in a head-to-head orientation separated by only 57 nt. This same organization is conserved at the human Rap1–KARS locus. When 5′ regions of the cRap1 and cKARS genes were tested for promoter activity, the promoters of both genes were found to lie in or near the intergenic spacer. The two promoters lack TATA boxes but appear to have downstream promoter elements (DPEs). Analysis of human Rap1 and KARS expressed sequence tags (ESTs) indicated that this localization of TATA-less promoters to the intergenic spacer is a conserved feature of the Rap1–KARS locus.

BRCA1 interacts with FHL2 and enhances FHL2 transactivation function

Germ-line mutations in BRCA1 are associated with an increased lifetime risk of developing breast and/or ovarian tumors. The BRCA1 gene product is a 220-kDa protein that contains a tandem of two BRCA1 C-terminal (BRCT) domains required for transcription. In an attempt to understand how BRCA1 exerts its function through BRCT domains, we search for partners of the BRCT domains of BRCA1. Using the yeast two-hybrid system, we identified the four and a half LIM-only protein 2 (FHL2) as a novel BRCA1 interacting protein. We demonstrate that BRCA1 and FHL2 can physically associate in vitro, in yeast, and in human cells. BRCA1 interacted with FHL2 through its second BRCT domain and the interaction of FHL2 with BRCA1 requires the last three LIM domains of FHL2. BRCA1 enhanced FHL2-mediated transcriptional activity in transient transfections. Tumor-derived transactivation-deficient BRCA1 mutants showed a reduced ability to enhance transactivation by FHL2. Lack of BRCA1 binding sites in the FHL2 completely abolished the FHL2 transactivation function. Reverse transcription polymerase chain reaction analysis showed that FHL2 mRNA levels may be downregulated in many breast cancer cell lines. These results suggest that the BRCA1–FHL2 interaction may be involved in transcriptional regulation and play a significant role in cancer cell growth.

The Arabidopsis MEI1 gene encodes a protein with five BRCT domains that is involved in meiosis-specific DNA repair events independent of SPO11-induced DSBs.

Arabidopsis thaliana MEI1 was first described as a gene involved in male meiosis, encoding a short protein showing homology with a human acrosin-trypsin inhibitor. We have isolated a new allele of mei1, and shown that in both mutants male and female meiosis are affected. In both reproductive pathways, meiosis proceeds while chromosomes become fragmented, resulting in aberrant meiotic products and in a strongly reduced fertility. We have shown that the gene mutated in mei1 mutants actually encodes a protein of 972 amino acids that contains five BRCA1 C-terminus (BRCT) domains and is similar to proteins involved in the response to DNA damage and replication blocks in eukaryotes. During meiosis, recombination is initiated by the formation of DNA double strand breaks (DSBs) induced by the protein SPO11. We analysed meiotic chromosome behaviour of the mei1 mutant in a spo11 mutant background and proved that the meiotic fragmentation observed in mei1 mutants was not the consequence of defects in the repair of meiotic DSBs induced by SPO11. We also analysed the effect of mei1 on the mitotic cell cycle but could not detect any sensitivity of mei1 seedlings to DNA-damaging agents like gamma-rays or UV. Therefore, MEI1 is a BRCT-domain-containing protein that could be specific to the meiotic cell cycle and that plays a crucial role in some DNA repair events independent of SPO11 DSB recombination repair.

From genotype to phenotype: correlating XRCC1 polymorphisms with mutagen sensitivity

This study correlated the extent of induced in vitro chromosomal damage, assessed by the mutagen sensitivity assay, with genotypes of the X-ray repair cross complementing group 1 ( XRCC1) gene, which encodes for a base excision repair protein. There are two common polymorphisms that cause amino acid substitutions in XRCC1, one at codon 194 in exon 6 and another at codon 399 in exon 10. We genotyped these two polymorphisms in 524 healthy subjects and performed mutagen sensitivity assays using both bleomycin and benzo[ a]pyrene-diol-epoxide (BPDE) as challenge mutagens. Our results showed that individuals with the wildtype exon 6 Arg/Arg exhibited significantly higher values of chromosomal breaks per cell (b/c) than those with one or two variant Trp alleles ( P=0.005 for bleomycin and P=0.05 for BPDE). For the exon 10 polymorphism, subjects who were Gln/Gln homozygotes had higher b/c than did those with other genotypes, with evidence of a gene dosage effect. When we combined the two polymorphic sites and used the exon 6 Arg/Trp and Trp/Trp and exon 10 Arg/Arg genotypes as the reference category, these differences were enhanced for bleomycin sensitivity ( P for trend = 0.032), but not for BPDE sensitivity ( P for trend = 0.821). These data are biologically plausible since codon 399 is located within the BRCA1 C-terminus functional domain and codon 194 is in the linker region of the XRCC1 N-terminal functional domain. To our knowledge, this is the largest study conducted evaluating the functional relevance of these polymorphisms.

BRCA1 associates with human papillomavirus type 18 E2 and stimulates E2-dependent transcription

BRCA1 is a breast and ovarian cancer-related tumor suppressor and has a role in transcriptional activation. We show here that BRCA1 enhances human papillomavirus type 18 (HPV-18) E2-dependent transcription in vivo. Using biochemical approaches, we discovered that BRCA1 interacts with the carboxyl-terminus of HPV-18 E2 protein in vivo and in vitro. Point mutations in the C-terminus make BRCA1 defective in transcriptional activation in E2-dependent transcription. This finding suggests that the C-terminus of BRCA1 is important for E2-dependent transcription. A chromatin immunoprecipitation assay indicated that BRCA1 is recruited onto the E2-dependent promoter region though E2 tethered to E2 binding sites in vivo. These results implicate that BRCA1 plays a role in E2-dependent transcription.

Role of BRCA1 in cellular resistance to paclitaxel and ionizing radiation in an ovarian cancer cell line carrying a defective BRCA1.

BRCA1, the gene responsible for approximately half of all cases of hereditary breast cancer and almost all cases of combined hereditary breast and ovarian cancer, has been implicated in the maintenance of genomic stability through DNA repair. This function is mediated, at least in part, through two tandem BRCA1 C-terminal (BRCT) repeats. The role of BRCA1 in the development of ovarian cancer is poorly understood, partially owing to the lack of ovarian cancer cell lines with defective BRCA1. The purpose of this study was to further characterize an endometrioid ovarian cancer cell line, SNU-251, which was previously reported to carry a nonsense mutation (from G to A) at amino acid 1815 of BRCA1. In addition, we examined the role of BRCA1 in the cell cycle and in the responses to the chemotherapy drug paclitaxel and ionizing radiation. Loss of the C-terminal 49 amino acids due to this point mutation did not affect the expression of the truncated BRCA1 protein, but caused a loss of transcriptional activation of the endogenous p21(WAF1/CIP1) gene, and could not sustain arrest in the G(2)/M phase of the cell cycle. The BRCA1 mutation in SNU-251 cells inhibited BRCA1 subnuclear assembly for DNA-damage repair and increased cellular sensitivity to ionizing radiation and paclitaxel. This sensitivity was reversed by reintroduction of ectopic wild-type BRCA1. Our results suggest that the deletion of the C-terminal 49 amino acids of BRCA1 results in a loss of BRCA1 function in the SNU-251 cell line. BRCA1 helps to mediate the resistance to both radiation and paclitaxel. Therefore, SNU-251 may be a useful model for studying the molecular mechanism of BRCA1 in the resistance of ovarian cancer to ionizing radiation and chemotherapy treatment and in the development of hereditary human ovarian cancer.

Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability.

DNA double-strand breaks represent the most potentially serious damage to a genome and hence, at least two pathways of DNA repair have evolved; namely, homologous recombination repair and non-homologous end joining. Defects in both rejoining processes result in genomic instability including chromosome rearrangements, LOH and gene mutations, which may lead to development of malignancies. Nijmegen breakage syndrome is a recessive genetic disorder, characterized by elevated sensitivity to ionizing radiation that induces double-strand breaks, and high frequency of malignancies. NBS1, the product of the gene underlying the disease, forms a multimeric complex with hMRE11/hRAD50 nuclease and recruits them to the vicinity of sites of DNA damage by direct binding to phosphorylated histone H2AX. The combination of the highly-conserved NBS1 forkhead associated domain and BRCA1 C-terminus domain has a crucial role for recognition of damaged sites. Thereafter, the NBS1-complex proceeds to rejoin double-strand breaks predominantly by homologous recombination repair in vertebrates. This process collaborates with cell-cycle checkpoints at S and G2 phase to facilitate DNA repair. NBS1 is also associated with telomere maintenance and DNA replication. Based on recent knowledge regarding NBS1, we propose here a two-step binding mechanism for damage recognition by repair proteins, and describe the molecular links to factors for genome stability.

Petasiphenol: a DNA polymerase lambda inhibitor.

Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicus, selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta, and epsilon but also showed no effect even on the pol beta activity, the three-dimensional structure of which is thought to be highly similar to pol lambda. The inhibitory effect of petasiphenol on intact pol lambda including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8 microM. The petasiphenol-induced inhibition of the pol lambda activity was noncompetitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol lambda including the pol beta-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol lambda but did not bind to the C-terminal region. On the basis of these results, the pol lambda inhibitory mechanism of petasiphenol is discussed.

BRCA1 interacts with acetyl-CoA carboxylase through its tandem of BRCT domains

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 is a 220-kDa protein that contains a tandem of two BRCA1 C-Terminal (BRCT) domains. Among missense and nonsense BRCA1 mutations responsible for family breast cancer, some are located into the BRCT tandem of BRCA1 coding sequence. In an attempt to understand how BRCT is critical for BRCA1 function, we search for partners of this BRCT tandem of BRCA1. Using a glutathione-S-transferase (GST) pull-down assay with murine cells, we isolated only one major BRCA1-interacting protein, further identified as Acetyl Coenzyme A (CoA) Carboxylase alpha (ACCA). We showed that this interaction is conserved through murine and human species. We also delineated the minimum interacting region as being the whole tandem of BRCT domains. We demonstrated that BRCA1 interacts in vitro and in vivo with ACCA. This interaction is completely abolished by five distinct germline BRCA1 deleterious mutations affecting the BRCT tandem of BRCA1. Interestingly, ACCA originally known as a rate-limiting enzyme for fatty acids biosynthesis, has been recently shown to be over-expressed in breast cancers and considered as a potential target for anti-neoplastic therapy. Furthermore, our observation is making a bridge between the genetic factors involved in susceptibility to breast and ovarian cancers, and environmental factors such as nutrition considered as key elements in the etiology of those cancers.

Mutations in the BRCT Domain Confer Temperature Sensitivity to BRCA1 in Transcription Activation

BRCA1 is a tumor suppressor gene and germ line mutations account for the majority of familial cases of breast and ovarian cancer. There is mounting evidence that BRCA1 functions in DNA repair and transcriptional regulation. A major hurdle to dissect the role of BRCA1 is the lack of molecular reagents to carry out biochemical and genetic experiments. Therefore, we used random mutagenesis of the C-terminus of BRCA1 (aa 1560–1863) to generate temperature-sensitive (TS) mutants in transcription activation. We obtained 11 TS mutants in transcription that localized primarily to the hydrophobic core of the BRCT-N domain of BRCA1. One of the mutants, H1686Q, also displayed temperature-dependent transcription activation in human cells. These conditional mutants represent valuable tools to assess the role of BRCA1 in transcription activation. Key Words:BRCA1, Yeast, Yranscription, Temperature- sensitive mutants, BRCT domain

Caspase-3 mediated cleavage of BRCA1 during UV-induced apoptosis.

The breast cancer suppressor protein, BRCA1 plays an important role in mediating cell cycle arrest, apoptosis and DNA responses to DNA damage signals. In this study, we show that BRCA1 level is downregulated during UV-induced apoptosis by caspase-3 mediated cleavage. Cleavage of BRCA1 by caspase-3 produced a fragment that contained the C-terminal of the molecule. Accordingly, treatment of cells with caspase-3 inhibitor or mutation of a specific caspase-3 cleavage site (DLLD) at amino acid 1151-1154 of BRCA1 abolished cleavage and consequential accumulation of the BRCA1 C-terminal fragment. Whereas expression of the non-cleavable BRCA1 (D/A 1154) mutant conferred the resistance phenotype to UV-induced cell death, expression of the cleaved BRCA1 C-terminus induced cell death in the absence of UV. Examination of the mechanism of C-terminus-induced cell death revealed that the cleaved fragment triggers the apoptotic response through activation of BRCA1 downstream effectors, GADD45 and JNK. Altogether, results of our study demonstrate a functional role for caspase-3 mediated cleavage of BRCA1 during UV-induced apoptosis.

Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor

The BRCT (BRCA1 C-terminus) is an evolutionary conserved protein-protein interacting module found as single, tandem or multiple repeats in a diverse range of proteins known to play roles in the DNA-damage response. The BRCT domains of 53BP1 bind to the tumour suppressor p53. To investigate the nature of this interaction, we have determined the crystal structure of the 53BP1 BRCT tandem repeat in complex with the DNA-binding domain of p53. The structure of the 53BP1-p53 complex shows that the BRCT tandem repeats pack together through a conserved interface that also involves the inter-domain linker. A comparison of the structure of the BRCT region of 53BP1 with the BRCA1 BRCT tandem repeat reveals that the interdomain interface and linker regions are remarkably well conserved. 53BP1 binds to p53 through contacts with the N-terminal BRCT repeat and the inter-BRCT linker. The p53 residues involved in this binding are mutated in cancer and are also important for DNA binding. We propose that BRCT domains bind to cellular target proteins through a conserved structural element termed the 'BRCT recognition motif'.