1H, 15N and 13C resonance backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA.

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 ß-sheets, closely resembling the typical structural topology of most BRCT domains.

BRCA1 and Its Vulnerable C-Terminal BRCT Domain: Structure, Function, Genetic Mutations and Links to Diagnosis and Treatment of Breast and Ovarian Cancer.

The BRCA1 is a tumor suppressor gene that encodes for the BRCA1 protein, which plays a vital role in DNA repair, cell cycle regulation, and the maintenance of genomic stability. The BRCA1 protein interacts with a variety of other proteins that play essential roles in gene regulation and embryonic development. It is a large protein composed of multiple domains. The C-terminal region of the BRCA1 protein consists of two BRCT domains connected by a short linker. The BRCT domains are crucial in protein-protein interactions as well as in DNA damage response and cell cycle regulation through their phosphoprotein binding modules that recognize the phosphorylated protein sequence motif of other kinases. Mutations within the BRCT domain can disrupt the normal function of BRCA1 and lead to an increased risk of developing breast and ovarian cancer. Herein, we explore the structural characteristics of BRCA1, focusing on the BRCT domain, its interactions with key cellular components, and its involvement in various cellular processes. In addition, the impact of BRCT domain mutations on breast and ovarian cancer susceptibility, prognosis, and treatment options is discussed. By providing a comprehensive understanding of the BRCT domain of BRCA1, this review aims to shed light on the role of this important domain in the pathogenesis and potential therapeutic approaches for breast and ovarian cancer.

Identification of plants' functional counterpart of the metazoan mediator of DNA Damage checkpoint 1.

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

NMR study of the structure and dynamics of the BRCT domain from the kinetochore protein KKT4.

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463-645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.

Inter-BRCT linker is probably the most intolerant region of the BRCA1 BRCT domain.

Pathogenic mutations in BRCA1 are associated with an increased risk of hereditary breast, ovarian, and some other cancers; however, the clinical significance of many mutations in this gene remains unknown (Variants of Unknown Significance/VUS). Since mutations in intolerant regions of a protein lead to dysfunction and pathogenicity, identifying these regions helps to predict the clinical importance of VUSs. This study aimed to identify intolerant regions of BRCA1 and understand the possible root of this susceptibility. Intolerant regions appear to carry more pathogenic mutations than expected due to their lower tolerance to missense variations. Therefore, we hypothesized that among the BRCA1 regions, the higher the mutation density, the greater the intolerance. Thus, pathogenic mutation density and regional intolerance scores were calculated to identify BRCA1-intolerant regions. To investigate the pathogenic mechanisms of missense-intolerant regions in BRCA1, transcription activation (TA) experiments and molecular dynamics (MD) simulations were also performed. The results showed that the RING domain, followed by the BRCT domain, has the highest density of pathogenic mutations. In the BRCT domain, a higher density of pathogenic mutations was observed in the inter-BRCT linker. Additionally, scores generated by Missense Tolerance Ratio-3D (MTR3D) and the Missense Tolerance Ratio consensus (MTRX) showed that the inter-BRCT linker is more intolerant than other regions of the BRCT domain. The MD results showed that mutations in the inter-BRCT linker led to cancer susceptibility, likely due to disruption of the interaction between BRCA1 and phosphopeptides. TA laboratory assays further supported the importance of the inter-BRCT linker.Communicated by Ramaswamy H. Sarma.

A small-molecule inhibitor of TopBP1 exerts anti-MYC activity and synergy with PARP inhibitors.

We have previously identified TopBP1 (topoisomerase IIß-binding protein 1) as a promising target for cancer therapy, given its role in the convergence of Rb, PI(3)K/Akt, and p53 pathways. Based on this, we conducted a large-scale molecular docking screening to identify a small-molecule inhibitor that specifically targets the BRCT7/8 domains of TopBP1, which we have named 5D4. Our studies show that 5D4 inhibits TopBP1 interactions with E2F1, mutant p53, and Cancerous Inhibitor of Protein Phosphatase 2A. This leads to the activation of E2F1-mediated apoptosis and the inhibition of mutant p53 gain of function. In addition, 5D4 disrupts the interaction of TopBP1 with MIZ1, which in turn allows MIZ1 to bind to its target gene promoters and repress MYC activity. Moreover, 5D4 inhibits the association of the TopBP1-PLK1 complex and prevents the formation of Rad51 foci. When combined with inhibitors of PARP1/2 or PARP14, 5D4 synergizes to effectively block cancer cell proliferation. Our animal studies have demonstrated the antitumor activity of 5D4 in breast and ovarian cancer xenograft models. Moreover, the effectiveness of 5D4 is further enhanced when combined with a PARP1/2 inhibitor talazoparib. Taken together, our findings strongly support the potential use of TopBP1-BRCT7/8 inhibitors as a targeted cancer therapy.

BRCA1 the Versatile Defender: Molecular to Environmental Perspectives.

The evolving history of BRCA1 research demonstrates the profound interconnectedness of a single protein within the web of crucial functions in human cells. Mutations in BRCA1, a tumor suppressor gene, have been linked to heightened breast and ovarian cancer risks. However, despite decades of extensive research, the mechanisms underlying BRCA1's contribution to tissue-specific tumor development remain elusive. Nevertheless, much of the BRCA1 protein's structure, function, and interactions has been elucidated. Individual regions of BRCA1 interact with numerous proteins to play roles in ubiquitination, transcription, cell checkpoints, and DNA damage repair. At a cellular scale, these BRCA1 functions coordinate tumor suppression, R-loop prevention, and cellular differentiation, all of which may contribute to BRCA1's role in cancer tissue specificity. As research on BRCA1 and breast cancer continues to evolve, it will become increasingly evident that modern materials such as Bisphenol A should be examined for their relationship with DNA stability, cancer incidence, and chemotherapy. Overall, this review offers a comprehensive understanding of BRCA1's many roles at a molecular, cellular, organismal, and environmental scale. We hope that the knowledge gathered here highlights both the necessity of BRCA1 research and the potential for novel strategies to prevent and treat cancer in individuals carrying BRCA1 mutations.

Structure/function studies of the NAD+-dependent DNA ligase from the poly-extremophile Deinococcus radiodurans reveal importance of the BRCT domain for DNA binding.

Bacterial NAD+-dependent DNA ligases (LigAs) are enzymes involved in replication, recombination, and DNA-repair processes by catalyzing the formation of phosphodiester bonds in the backbone of DNA. These multidomain proteins exhibit four modular domains, that are highly conserved across species, with the BRCT (breast cancer type 1 C-terminus) domain on the C-terminus of the enzyme. In this study, we expressed and purified both recombinant full-length and a C-terminally truncated LigA from Deinococcus radiodurans (DrLigA and DrLigA∆BRCT) and characterized them using biochemical and X-ray crystallography techniques. Using seeds of DrLigA spherulites, we obtained ≤ 100 µm plate crystals of DrLigA∆BRCT. The crystal structure of the truncated protein was obtained at 3.4 Å resolution, revealing DrLigA∆BRCT in a non-adenylated state. Using molecular beacon-based activity assays, we demonstrated that DNA ligation via nick sealing remains unaffected in the truncated DrLigA∆BRCT. However, DNA-binding assays revealed a reduction in the affinity of DrLigA∆BRCT for dsDNA. Thus, we conclude that the flexible BRCT domain, while not critical for DNA nick-joining, plays a role in the DNA binding process, which may be a conserved function of the BRCT domain in LigA-type DNA ligases.

BRCA1/TP53 tumor proteins inhibited by novel analogues of curcumin - Insight from computational modelling, dynamic simulation and experimental validation.

The current study aimed to design novel curcumin analogue inhibitors with antiproliferative and antitumor activity towards BRCA1 and TP53 tumor proteins and to study their therapeutic potential by computer-aided molecular designing and experimental investigations. Four curcumin analogues were computationally designed and their drug-likeness and pharmacokinetic properties were predicted. The binding of these analogues against six protein targets belonging to BRCA1 and TP53 tumor proteins were modelled by molecular docking and their binding energies were compared with that of curcumin and the standard drug cyclophosphamide and its validated target. The stabilities of selected docked complexes were confirmed by molecular dynamic simulation (MDS) and MMGBSA calculations. The best-docked analogue was chemically synthesized, characterized, and used for in vitro cytotoxic screening using DLA, EAC, and C127I cell lines. In vivo antitumor studies were carried out in Swiss Albino Mice. The study revealed that the designed analogues satisfied drug-likeness and pharmacokinetic properties and demonstrated better binding affinity to the selected targets than curcumin. Among the analogues, NLH demonstrated significant interaction with the BRCA1-BRCT-c domain (TG3; binding energy -8.3 kcal/mol) when compared to the interaction of curcumin (binding energy -6.19 kcal) and cyclophosphamide (binding energy -3.8 kcal/mol) and its usual substrate (TG7). The MDS and MM/GBSA studies revealed that the binding free energy of the NLH-TG3 complex (-61.24 kcal/mol) was better when compared to that of the cyclophosphamide-TG7 complex (-21.67 kcal/mol). In vitro, cytotoxic studies showed that NLH demonstrated significant antiproliferative activities against tumor cell lines. The in vivo study depicted NLH possesses the potential for tumor inhibition. Thus, the newly synthesized curcumin analogue is probably used to develop novel therapeutic agents against breast cancer.

Evolution of the triplet BRCT domain.

Organisms have evolved a complex system, called the DNA damage response (DDR), which maintains genome integrity. The DDR is responsible for identifying and repairing a variety of lesions and alterations in DNA. DDR proteins coordinate DNA damage detection, cell cycle arrest, and repair, with many of these events regulated by protein phosphorylation. In the human proteome, 23 proteins contain the BRCT (BRCA1 C-Terminus domain) domain, a modular signaling domain that can bind phosphopeptides and mediate protein-protein interactions. BRCTs can be found as functional single units, tandem (tBRCT), triplet (tpBRCT), and quartet. Here we examine the evolution of the tpBRCT architecture present in TOPBP1 (DNA topoisomerase II binding protein 1) and ECT2 (epithelial cell transforming 2), and their respective interaction partners RAD9 (Cell cycle checkpoint control protein RAD9) and CYK-4 (Rac GTPase-activating protein 1), with a focus on the conservation of the phosphopeptide-binding residues. The pair TOPBP1-RAD9 arose with the Eukaryotes and ECT2-CYK-4 with the Eumetazoans. Triplet structural and functional characteristics were conserved in almost all organisms. The first unit of the triplet (BRCT0) is different from the other two BRCTs but conserved between orthologs for both TOPBP1 and ECT2. BRCT domain evolution simulations suggest a trend to retain the singlet or towards two or three BRCT copies per protein consistent with functional tBRCT and tpBRCT architectures. Our results shed light on the emergence of the function and architecture of multiple BRCT domain organizations and provide information about the evolution of the BRCT triplet. Knowledge of BRCT domain evolution can improve the understanding of DNA damage response mechanisms and signal transduction in DDR.

Structural dynamics of clinically-reported VUS in the BARD1 ARD-BRCT region to predict the molecular basis of alterations.

The functional domains of BARD1, comprise the Ankyrin Repeat Domain (ARD), C-Terminal domains (BRCTs), and a linker between ARD and the BRCTs, which are known to bind to Cleavage stimulation Factor complex-subunit of 50 kDa (CstF-50). The pathogenic mutation Q564H in the BARD1 ARD-linker-BRCT region has been reported to abrogate the binding between BARD1 and CstF-50. Intermediate penetrance variants of BARD1 are associated with the occurrence of breast cancer. Therefore, seven missense variants of unknown significance (VUS), L447V, P454L, N470S, V507M, I509T, C557S, and Q564H of BARD1, reported in the ARD domain and the linker region were evaluated via molecular dynamics (MD) simulations. The mutants revealed statistically significantly different distributions of RMSD (root mean square deviation), residuewise RMSF (root mean square fluctuation), Rg (radius of gyration), SASA (solvent accessible surface area), and COM (centre of mass)-to-COM distance between the ARD and the BRCT repeat, between the wild type and each mutant. The secondary structural composition of the mutants was slightly altered relative to that of the wild type. However, the reported in-silico based prediction require further validation using in-vitro, biophysical and structure-based approachCommunicated by Ramaswamy H. Sarma.

BRCT Domains: Structure, Functions, and Implications in Disease-New Therapeutic Targets for Innovative Drug Discovery against Infections.

The search for new therapeutic targets and their implications in drug development remains an emerging scientific topic. BRCT-bearing proteins are found in Archaea, Bacteria, Eukarya, and viruses. They are traditionally involved in DNA repair, recombination, and cell cycle control. To carry out these functions, BRCT domains are able to interact with DNA and proteins. Moreover, such domains are also implicated in several pathogenic processes and malignancies including breast, ovarian, and lung cancer. Although these domains exhibit moderately conserved folding, their sequences show very low conservation. Interestingly, sequence variations among species are considered positive traits in the search for suitable therapeutic targets, since non-specific drug interactions might be reduced. These main characteristics of BRCT, as well as its critical implications in key biological processes in the cell, have prompted the study of these domains as therapeutic targets. This review explores the possible roles of BRCT domains as therapeutic targets for drug discovery. We describe their common structural features and relevant interactions and pathways, as well as their implications in pathologic processes. Drugs commonly used to target these domains are also presented. Finally, based on their structures, we describe new drug design possibilities using modern and innovative techniques.

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

The TOPBP1 and NBS1 proteins are key components of DNA repair and DNA-based signaling systems. TOPBP1 is a multi-BRCT domain containing protein that plays important roles in checkpoint signaling, DNA replication, and DNA repair. Likewise, NBS1, which is a component of the MRE11-RAD50-NBS1 (MRN) complex, functions in both checkpoint signaling and DNA repair. NBS1 also contains BRCT domains, and previous works have shown that TOPBP1 and NBS1 interact with one another. In this work we examine the interaction between TOPBP1 and NBS1 in detail. We report that NBS1 uses its BRCT1 domain to interact with TOPBP1's BRCT1 domain and, separately, with TOPBP1's BRCT2 domain. Thus, NBS1 can make two distinct contacts with TOPBP1. We report that recombinant TOPBP1 and NBS1 proteins bind one another in a purified system, showing that the interaction is direct and does not require post-translational modifications. Surprisingly, we also report that intact BRCT domains are not required for these interactions, as truncated versions of the domains are sufficient to confer binding. For TOPBP1, we find that small 24-29 amino acid sequences within BRCT1 or BRCT2 allow binding to NBS1, in a transferrable manner. These data expand our knowledge of how the crucial DNA damage response proteins TOPBP1 and NBS1 interact with one another and set the stage for functional analysis of the two disparate binding sites for NBS1 on TOPBP1.

The multi-BRCT domain protein DDRM2 promotes the recruitment of RAD51 to DNA damage sites to facilitate homologous recombination.

DNA double-strand breaks (DSBs) are the most toxic form of DNA damage in cells. Homologous recombination (HR) is an error-free repair mechanism for DSBs as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. Through genetic screens for DNA damage response mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including bryophytes, the earliest land plant lineage. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. In support, genetic analysis suggests that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Mechanistically, DDRM2 interacts with the core HR protein RAD51 and is required for the recruitment of RAD51 to DSB sites. Our study reveals that SOG1-DDRM2-RAD51 is a novel module for HR, providing a potential target for improving the efficiency of gene targeting.

Bractoppin, a BRCA1 carboxy-terminal domain (BRCT) inhibitor, suppresses tumor progression in ovarian borderline tumor organoids.

Borderline ovarian tumors are a special class of ovarian tumors between benign and malignant, which are not sensitive to traditional chemotherapy regimens, and the development of target drugs is limited due to the lack of cell lines. Tumor organoids can well preserve the genetic characteristics of the primary tumor, but there are only a few reports of application in borderline tumors. In this study, we successfully generated 13 ovarian borderline tumor organoids and tested the antitumor activity of Bractoppin, a BRCA1 carboxy-terminal domain (BRCT) inhibitor. Bractoppin promotes organoid apoptosis. Mechanistically, Bractoppin can inhibit organoid cell cycle progression, inhibit the repair of DSB damage and promote tumor cell apoptosis. In addition, Bractoppin can also promote the apoptosis of ovarian cancer cell lines and inhibit the HR and NHEJ repair ability of tumor cells. We demonstrate the value of ovarian borderline tumor organoids in the exploration of molecular therapy drugs, and Bractoppin may be a valuable small molecule drug in the treatment of BOT.

Analysis of BRCT5 domain-containing proteins reveals a new component of DNA damage repair in Arabidopsis.

The integrity of plant genetic information is constantly challenged by various internal and external factors. Therefore, plants use a sophisticated molecular network to identify, signal and repair damaged DNA. Here, we report on the identification and analysis of four uncharacterized Arabidopsis BRCT5 DOMAIN CONTAINING PROTEINs (BCPs). Proteins with the BRCT5 domain are frequently involved in the maintenance of genome stability across eukaryotes. The screening for sensitivity to induced DNA damage identified BCP1 as the most interesting candidate. We show that BCP1 loss of function mutants are hypersensitive to various types of DNA damage and accumulate an increased number of dead cells in root apical meristems upon DNA damage. Analysis of publicly available sog1 transcriptomic and SOG1 genome-wide DNA binding data revealed that BCP1 is inducible by gamma radiation and is a direct target of this key DNA damage signaling transcription factor. Importantly, bcp1 plants showed a reduced frequency of somatic homologous recombination in response to both endogenous and induced DNA damage. Altogether, we identified a novel plant-specific DNA repair factor that acts downstream of SOG1 in homology-based repair.

The BRCT Domain from the Homologue of the Oncogene PES1 in Leishmania major (LmjPES) Promotes Malignancy and Drug Resistance in Mammalian Cells.

Around 15% of cancer cases are attributable to infectious agents. Epidemiological studies suggest that an association between leishmaniasis and cancer does exist. Recently, the homologue of PES1 in Leishmania major (LmjPES) was described to be involved in parasite infectivity. Mammalian PES1 protein has been implicated in cellular processes like cell cycle regulation. Its BRCT domain has been identified as a key factor in DNA damage-responsive checkpoints. This work aimed to elucidate the hypothetical oncogenic implication of BRCT domain from LmjPES in host cells. We generated a lentivirus carrying this BRCT domain sequence (lentiBRCT) and a lentivirus expressing the luciferase protein (lentiLuc), as control. Then, HEK293T and NIH/3T3 mammalian cells were infected with these lentiviruses. We observed that the expression of BRCT domain from LmjPES conferred to mammal cells in vitro a greater replication rate and higher survival. In in vivo experiments, we observed faster tumor growth in mice inoculated with lentiBRCT respect to lentiLuc HEK293T infected cells. Moreover, the lentiBRCT infected cells were less sensitive to the genotoxic drugs. Accordingly, gene expression profiling analysis revealed that BRCT domain from LmjPES protein altered the expression of proliferation- (DTX3L, CPA4, BHLHE41, BMP2, DHRS2, S100A1 and PARP9), survival- (BMP2 and CARD9) and chemoresistance-related genes (DPYD, Dok3, DTX3L, PARP9 and DHRS2). Altogether, our results reinforced the idea that in eukaryotes, horizontal gene transfer might be also achieved by parasitism like Leishmania infection driving therefore to some crucial biological changes such as proliferation and drug resistance.

Decoding histone ubiquitylation.

Histone ubiquitylation is a critical part of both active and repressed transcriptional states, and lies at the heart of DNA damage repair signaling. The histone residues targeted for ubiquitylation are often highly conserved through evolution, and extensive functional studies of the enzymes that catalyze the ubiquitylation and de-ubiquitylation of histones have revealed key roles linked to cell growth and division, development, and disease in model systems ranging from yeast to human cells. Nonetheless, the downstream consequences of these modifications have only recently begun to be appreciated on a molecular level. Here we review the structure and function of proteins that act as effectors or "readers" of histone ubiquitylation. We highlight lessons learned about how ubiquitin recognition lends specificity and function to intermolecular interactions in the context of transcription and DNA repair, as well as what this might mean for how we think about histone modifications more broadly.

Delineation of a minimal topoisomerase II binding protein 1 for regulated activation of ATR at DNA double-strand breaks.

Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangiectasia and Rad3-related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal repeat (BRCT) motif, which allows protein-protein and protein-DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-interacting protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1's nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here, we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks in a regulated manner. We named this minimal TOPBP1 "Junior" and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT7&8. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DNA double-strand breaks and is dispensable thereafter, and that BRCT7&8 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1 and provides insight into the mechanism of ATR activation.

BARD1 is an ATPase activating protein for OLA1.

OLA1 is a P-loop ATPase, implicated in centrosome duplication through the interactions with tumor suppressors BRCA1 and BARD1. Disruption of the interaction of OLA1 with BARD1 results in centrosome amplification. However, the molecular interplay and mechanism of the OLA1-BARD1 complex remain elusive. Here, we use a battery of biophysical, biochemical, and structural analyses to elucidate the molecular basis of the OLA1-BARD1 interaction. Our structural and enzyme kinetics analyses show this nucleotide-dependent interaction enhances the ATPase activity of OLA1 by increasing the turnover number (kcat). Unlike canonical GTPase activating proteins that act directly on the catalytic G domain, the BARD1 BRCT domain binds to the OLA1 TGS domain via a highly conserved BUDR motif. A cancer related mutation V695L on BARD1 is known to associate with centrosome abnormality. The V695L mutation reduces the BARD1 BRCT-mediated activation of OLA1. Crystallographic snapshot of the BRCT V695L mutant at 1.88 Å reveals this mutation perturbs the OLA1 binding site, resulting in reduced interaction. Altogether, our findings suggest the BARD1 BRCT domain serves as an ATPase activating protein to control OLA1 allosterically.