BRCT Domain Research Articles

CSB interacts with BRCA1 in late S/G2 to promote MRN- and CtIP-mediated DNA end resection.

CSB, a member of the SWI2/SNF2 superfamily, has been implicated in evicting histones to promote the DSB pathway choice towards homologous recombination (HR) repair. However, how CSB promotes HR repair remains poorly characterized. Here we demonstrate that CSB interacts with both MRE11/RAD50/NBS1 (MRN) and BRCA1 in a cell cycle regulated manner, with the former requiring its WHD and occurring predominantly in early S phase. CSB interacts with the BRCT domain of BRCA1 and this interaction is regulated by CDK-dependent phosphorylation of CSB on S1276. The CSB–BRCA1 interaction, which peaks in late S/G2 phase, is responsible for mediating the interaction of CSB with the BRCA1-C complex consisting of BRCA1, MRN and CtIP. While dispensable for histone eviction at DSBs, CSB phosphorylation on S1276 is necessary to promote efficient MRN- and CtIP-mediated DNA end resection, thereby restricting NHEJ and enforcing the DSB repair pathway choice to HR. CSB phosphorylation on S1276 is also necessary to support cell survival in response to DNA damage-inducing agents. These results altogether suggest that CSB interacts with BRCA1 to promote DNA end resection for HR repair and that although prerequisite, CSB-mediated histone eviction alone is insufficient to promote the pathway choice towards HR.

Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein-Protein Interactions of BRCA1 tBRCT Domain.

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure-activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein-protein interactions.

Molecular Basis for Control of Diverse Genome Stability Factors by the Multi-BRCT Scaffold Rtt107.

BRCT domains support myriad protein-protein interactions involved in genome maintenance. Although di-BRCT recognition of phospho-proteins is well known to support the genotoxic response, whether multi-BRCT domains can acquire distinct structures and functions is unclear. Here we present the tetra-BRCT structures from the conserved yeast protein Rtt107 in free and ligand-bound forms. The four BRCT repeats fold into a tetrahedral structure that recognizes unmodified ligands using a bi-partite mechanism, suggesting repeat origami enabling function acquisition. Functional studies show that Rtt107 binding of partner proteins of diverse activities promotes genome replication and stability in both distinct and concerted manners. A unified theme is that tetra- and di-BRCT domains of Rtt107 collaborate to recruit partner proteins to chromatin. Our work thus illustrates how a master regulator uses two types of BRCT domains to recognize distinct genome factors and direct them to chromatin for constitutive genome protection.

Abstract 3504: Mutation analysis of ovarian carcinoma patients presenting optimal response to neoadjuvant chemotherapy

Abstract Background: Neoadjuvant chemotherapy (NAC) followed by interval debulking does not present inferior results to those of primary cytoreduction and offers the opportunity to evaluate chemo-sensitivity in vivo. Pathological response scores (CRS) have been shown to correlate with outcome with a complete or near-complete CRS (CRS3) predicting improved progression-free survival. Approximately 20% of ovarian cancers present BRCA1/2 mutations, which predict a better response to platinum salts. Our proposal is to determine the prevalence of BRCA mutations or other homologous recombination (HR) alterations among patients with a CRS3 after NAC. Methods: We performed a retrospective analysis of clinical, pathological, and sequencing data of patients who experienced a complete or near-complete response to platinum based NAC. For patients with wild-type (WT) sequencing and with tumor samples available, somatic analysis based on Next Generation Sequencing (NGS) with a large panel of genes (BRCA1, BRCA2, ATM, BARD1, BRIP1, CCNE1, CDK12, CHEK2, PALB2,RAD51C, RAD51D, TP53), including specific non-coding regulatory regions of BRCA1, BRCA2 and RAD51C, was also performed. We present preliminary data on the 1st cohort analysed at the time of abstract submission. Results: A total of 33 patients were identified who demonstrated CRS3 post-NAC. Tumors were in majority EC IIIc (57%) and grade II-III serous ovarian carcinoma (90%). Median overall survival (m OS) of the entire cohort was 55 months (IC95% 36-74). To date, germline and/or somatic analyses were available on 23 pts. The prevalence of pathogenic BRCA1/2 variants is higher than expected in this cohort of patients presenting a CRS3. In total, 7 of 23 patients (30%) had a germline (4) or somatic pathogenic (3) BRCA variant. In addition, among the 16 BRCA1/2 WT pts subjected to further HR NGS analysis, the following cases of alterations were identified: one ATM nonsense mutation (c.2465T>G; p.Leu822*), one pathogenic mutation of CDK12 gene (c.3G>A;p.Met1?), one likely pathogenic variant located on BRCT domain of BRCA1 (c.5165C>T p.Ser1722Phe), two 3'UTR BRCA2 variants of uncertain significance (VUS) (c.*14C>T; c.*72A>G), and one BRIP1 VUS (c.2932G>C, p.Gly978Arg). Further, one BRCA WT case presented an unexpected CCNE1 amplification. Conclusion: HGOC patients presenting with a pCR are enriched for BRCA germline and/or somatic BRCA mutations. In addition, among the small subset of BRCA WT pCR tumors, a deleterious ATM and CDK12 mutation were identified. This prevalence can be underestimated in a context of pCR since somatic screening is impossible on the debulking material. Somatic BRCA1/2 and complete HR sequencing on the initial biopsy is recommended to identify additional pts with exclusive somatic mutations. More complete data on this cohort will be presented. Citation Format: Etienne Rouleau, Elizabeth Santana dos Santos, Francilette Maela, Ludovic Lacroix, Aurélie Auguste, Patricia Pautier, Philippe Morice, Catherine Genestie, Alexandra Leary. Mutation analysis of ovarian carcinoma patients presenting optimal response to neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3504.

Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint.

Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint.

Overexpression of BRCA1 in Neural Stem Cells Enhances Cell Survival and Functional Recovery after Transplantation into Experimental Ischemic Stroke.

Transplantation of neural stem cells (NSCs) is a promising therapy for ischemic stroke. However, the effectiveness of this approach is limited by grafted cell death. Breast cancer susceptibility protein 1 (BRCA1) could suppress apoptosis in neural progenitors and modulate oxidative stress in neurons. In this study, we found that BRCA1 was upregulated by oxygen-glucose deprivation/reoxygenation (OGD/R). Overexpression of BRCA1 in NSCs reduced cell apoptosis and oxidative stress after OGD/R insult. The molecule overexpression also stimulated cellular proliferation in OGD/R NSCs and increased the survival rate of grafted cells. Further, the transplantation of BRCA1-transfected NSCs into mice with ischemic stroke increased brain-derived neurotropic factor and nerve growth factor expression in the brain and elicited neurological function improvement. In addition, we found that RING finger domain and BRCT domain of BRCA1 could physically interact with p53 in NSCs. The cross talk between BRCA1 RING finger domain and p53 was responsible for p53 ubiquitination and degradation. Our findings indicate that modification with BRCA1 could enhance the efficacy of NSCs transplantation in ischemic stroke.

Exploring the Structural Link Between BRCA1 and Carcinogenesis

The breast cancer type 1 susceptibility gene (BRCA1), located on chromosome 17, codes for a tumor suppressor protein that functions as a caretaker gene in preserving chromosomal stability. The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationship between this protein, BRCA1, and breast cancer. BRCA1 functions in both checkpoint activation and double stranded DNA repair. Most of BRCA1's functions occur within the nucleus where it interacts with several other proteins to mend breaks in DNA. Over 1,800 mutations of BRCA1 have been discovered within the gene itself. Many of these mutations cause a truncated protein to be coded for, resulting in a malfunctional protein incapable of repairing damaged DNA. Another function of BRCA1 is to attach ubiquitin to other proteins, which helps to regulate processes in the body and is needed for homologous recombination. Without this type of DNA repair, cells can develop mutations leading to carcinogenesis. Other BRCA1 mutations trigger cells to grow and divide uncontrollably which can also result in carcinogenesis, specifically of breast cancer tumors. It is for this reason that BRCA1 gets the name breast cancer type 1 susceptibility protein. BRCA1 is a large phosphoprotein composed of 1,863 amino acids containing a zinc finger N‐terminal RING domain and two C‐terminals referred to as BRCT. The presence of a DNA‐binding, N‐terminal RING finger domain suggests that BRCA1 may play an undefined role in transcription regulation. RING mutations in BRCA1 have often been associated with familial carcinomas. The two BRCT regions of BRCA1 each have 90–100 amino acid sequence repeats called BRCT repeats. These repeats are typically associated with members of a protein fold family that includes many other proteins associated with DNA repair. Although the molecular basis of their effects remains largely obscure, many mutations are known to target the highly conserved C‐terminal BRCT repeats that function as a phosphoserine phosphothreonine‐binding module. It is hypothesized that if the structure of the BRCT‐BRCT interface is changed, the whole BRCT domain becomes defective. Future research and observation of the effects of other missense mutations on BRCA1 cellular function would be necessary to correlate BRCT loss of structure with BRCA1 loss of function. This would also further establish protein misfolding as a basic molecular mechanism of disease. Other research prospects include the potential of using BRCA1 mutations in order to predict treatment resistance in breast cancer, and using CRISPR gene therapies to edit mutated BRCA1 genes, therefore preventing or halting further carcinogenesis.Support or Funding InformationSponsors: Chris Elniff and L.B. FogtMentor: Dr. Jack Treml PhD, University of Kansas BiotechnologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of amino acid substitutions at secondary structures in the BRCT domains of the tumor suppressor BRCA1: Implications for clinical annotation

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.

Functional analysis of BARD1 missense variants in homology-directed repair and damage sensitivity.

The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants.

The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors

Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.

Genetic association and functional characterization of MCPH1 gene variation in bipolar disorder and schizophrenia.

A rare microcephalin 1 gene (MCPH1) variant rs61749465A>G (p.Asp61Gly) with prior evidence for association with schizophrenia (p = 3.78 × 10-7 ) was tested for association in 2,300 bipolar disorder (BPD) participants, 1,930 SCZ participants and 1,820 normal comparison subjects. We report evidence for association of rs61749465A>G with BPD (p = 0.0009). rs61749465 is located in the N-terminal of the BRCT1 domain of MCPH1. Bioinformatic analysis predicted the Asp61Gly substitution to be damaging to MCPH1 function. A second MCPH1 BRCT1 domain variant (rs199422124C>G; p.Thr27Arg), reported to cause autosomal recessive microcephaly, was not detected in the participants tested here. We sought to characterize the functional effects of these variants on MCPH1 function. Cell count assays indicated that rs199422124 allele G had a greater impact on cell survival compared to the G allele of rs61749465. Gene expression analysis combined with gene network and pathway analysis indicated that rs61749465 allele G may impact protein translation and cell cycle control. The evidence for association between rs61749465A>G and psychosis in both BPD and SCZ warrants further replication. Likewise, the data from the functional analyses point to molecular mechanisms that may underlie the proposed MCPH1 mediated risk of psychosis and pathogenesis in autosomal recessive microcephaly require additional experimental validation.

Identification of Novel Interaction Partners of Ets-1: Focus on DNA Repair

The transcription factor Ets-1 (ETS proto-oncogene 1) shows low expression levels except in specific biological processes like haematopoiesis or angiogenesis. Elevated levels of expression are observed in tumor progression, resulting in Ets-1 being named an oncoprotein. It has recently been shown that Ets-1 interacts with two DNA repair enzymes, PARP-1 (poly(ADP-ribose) polymerase 1) and DNA-PK (DNA-dependent protein kinase), through two different domains and that these interactions play a role in cancer. Considering that Ets-1 can bind to distinctly different domains of two DNA repair enzymes, we hypothesized that the interaction can be transposed onto homologs of the respective domains. We have searched for sequence and structure homologs of the interacting ETS(Ets-1), BRCT(PARP-1) and SAP(DNA-PK) domains, and have identified several candidate binding pairs that are currently not annotated as such. Many of the Ets-1 partners are associated to DNA repair mechanisms. We have applied protein-protein docking to establish putative interaction poses and investigated these using centrality analyses at the protein residue level. Most of the identified poses are virtually similar to our recently established interaction model for Ets-1/PARP-1 and Ets-1/DNA-PK. Our work illustrates the potentially high number of interactors of Ets-1, in particular involved in DNA repair mechanisms, which shows the oncoprotein as a potential important regulator of the mechanism.

Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

Repair of two major forms of DNA damage, single strand breaks and base modifications, are dependent on XRCC1. XRCC1 orchestrates these repair processes by temporally and spatially coordinating interactions between several other repair proteins. Here we show that XRCC1 contains a central DNA binding domain (CDB, residues 219–415) encompassing its first BRCT domain. In contrast to the N-terminal domain of XRCC1, which has been reported to mediate damage sensing in vitro, we demonstrate that the DNA binding module identified here lacks binding specificity towards DNA containing nicks or gaps. Alanine substitution of residues within the CDB of XRCC1 disrupt DNA binding in vitro and lead to a significant reduction in XRCC1 retention at DNA damage sites without affecting initial recruitment. Interestingly, reduced retention at sites of DNA damage is associated with an increased rate of repair. These findings suggest that DNA binding activity of XRCC1 plays a significant role in retention at sites of damage and the rate at which damage is repaired.

NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation.

DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation-dependent mode through NBS1 and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection occurs invivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11-RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

Combining Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of BRCA1 BRCT Variants on Cancer Risk.

BRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented on the basis of mutated BRCT domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis. IMPLICATIONS: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection.

Effect of single amino acid mutations on C-terminal domain of breast cancer susceptible protein 1

The most commonly diagnosed cancer in women is the breast cancer. Around 5-10% of breast cancer cases are hereditary, mainly due to the mutation in the breast cancer susceptible Breast Cancer 1 (BRCA1) and Breast Cancer 2 (BRCA2) tumour-suppressor genes. More than hundreds mutations are documented in BRCA1 C-terminal region (BRCT), mainly associated with repairing DNA damage and cell cycle control. In this study, we employed different mutation analysis system such as sorting intolerant from tolerant, MutPred, PON-P2, Meta-SNP, etc., to predict the pathological effects of 95 distinct miss sense mutation on BRCT domain. Out of which, 37 mutations were predicted to be deleterious by all mutation analysis systems affecting the protein stability and its normal function leading to causing cancer. The computational approach for finding out the impact of mutation on BRCA protein may provide a way in early detection and therapy in breast cancer patients.

Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1

SummaryXRCC1 accelerates repair of DNA single-strand breaks by acting as a scaffold protein for the recruitment of Polβ, LigIIIα, and end-processing factors, such as PNKP and APTX. XRCC1 itself is recruited to DNA damage through interaction of its central BRCT domain with poly(ADP-ribose) chains generated by PARP1 or PARP2. XRCC1 is believed to interact directly with DNA at sites of damage, but the molecular basis for this interaction within XRCC1 remains unclear. We now show that the central BRCT domain simultaneously mediates interaction of XRCC1 with poly(ADP-ribose) and DNA, through separate and non-overlapping binding sites on opposite faces of the domain. Mutation of residues within the DNA binding site, which includes the site of a common disease-associated human polymorphism, affects DNA binding of this XRCC1 domain in vitro and impairs XRCC1 recruitment and retention at DNA damage and repair of single-strand breaks in vivo.

M23 - GENETIC ASSOCIATION AND FUNCTIONAL CHARACTERIZATION OF A VARIANT IN THE MCPH1 GENE IN BIPOLAR DISORDER AND SCHIZOPHRENIA

Background Both Schizophrenia (SCZ) and Bipolar Disorder (BPD) disorder are serious psychiatric conditions that can give rise to debilitating symptoms that can severely impact the life of the affected individual, and their families. We have previously reported that the MCPH1 gene variant rs61749465 A>G (p.Asp61Gly) showed suggestive evidence for association with schizophrenia (SCZ; Leonenko et al. 2017). The MCPH1 gene has multiple roles in cellular functions including, DNA damage/repair pathways; centrosomal localization; E2F transcription factor 1-mediated apoptosis; transcriptional activation of cell cycle checkpoint, DNA stability; and telomere structure. A second MCPH1 point mutation (rs199422124 C>G; p.Thr27Arg) is an autosomal recessive cause of microcephaly. Methods The schizophrenia associated MCPH1 variant rs61749465 was tested for association in 2,300 Bipolar Disorder (BPD) subjects, and 1,820 normal comparison subjects. We also analysed the microcephaly causing mutation rs199422124 in the BPD and control subjects and in our SCZ cohort of 1,930 subjects. Next, we conducted analysis of the in vitro effects of the rs6174965 and rs199422124 variants on cell survival/proliferation using cell counts, and the MTT assay; their effect on DNA damage using the comet assay; their effect on mRNA stability using a combination of Actinomycin D treatment followed by Q-PCR; and their effect on gene expression using RNA-Seq analysis combined with pathway analysis and gene network analyses. Results After genotyping we report evidence for association with BPD (P=0.0009). Notably the variant allele of rs61749465 was absent in the 1,820 comparison subjects tested. rs61749465 is located in the N-terminal of the BRCT1 domain of MCPH1. Bioinformatic analysis predicted the Asp61Gly substitution to be damaging to MCPH1 protein function. rs199422124 was not detected in any of the participants. We sought to characterize the functional effects of these variants on MCPH1 function. Cell viability and cell count assays indicated that the variant allele of rs199422124 had a larger impact on cell survival compared to the variant allele of rs61749465, however neither of the variant alleles significantly altered DNA damage or mRNA stability. Gene expression analysis for rs61749465 using RNA-seq shows that the expression of a number of heat shock protein have been affected. Gene network and pathway analysis indicated that the variant alleles may impact cellular aging and protein translation. Discussion Further in vitro and in vivo characterization of the rs61749465 variant in MCPH1 may provide insight into the molecular pathogenesis of psychosis.

BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands.

TOPBP1 and its fission yeast homologueRad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 with diverse phospho-ligands, including human RAD9 and Treslin, and Schizosaccharomyces pombe Crb2 and Sld3, to define the determinants of BRCT domain specificity. We use this to identify and characterise previously unknown phosphorylation-dependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins.