BRCA1-associated Genome Surveillance Complex Research Articles

Involvement of Variants in the Genes Encoding BRCA1-Associated Genome Surveillance Complex (BASC) in the Development of Human Common Diseases

Involvement of Variants in the Genes Encoding BRCA1-Associated Genome Surveillance Complex (BASC) in the Development of Human Common Diseases

Вовлеченность генов белков BRСA1-ассоциированного комплекса наблюдения за геномом (BASC) в развитие многофакторной патологии

The code hypothesis postulates a relationship between Mendelian (monogenic) and common pathologies. In this hypothesis, polymorphisms in the genes of Mendelian diseases may have a significant contribution to predisposition to common diseases in which the same biochemical pathways may be involved. In this review a group of genes encoding various proteins participating in the DNA repair, with a particular focus on the BRCA1-associated genome surveillance complex (BASC), is presented through the prism of the code hypothesis. Here we discuss (1) their main functions in the repair of DNA double-strand breaks (ATM, MRE11, NBN, RAD50, BRCA1, and BLM) and mismatch repair (MSH2, MSH6, MLH1, PMS2, RF-C, and PCNA); (2) the mitochondrial involvement of these proteins; (3) the involvement of BASC proteins in the development of an adaptive immune response. For 13 out of 16 BASC protein encoding genes, mutations leading to monogenic diseases have already been described; for 11, there are associations with common diseases or individual biological processes. Patients with mutations in the genes of the BASC complex and patients with severe combined immunodeficiency share similar symptoms. Polymorphisms within DNA repair genes may play a role in the development of common diseases through the involvement of the immune response. The pleiotropic effects of these genes suggest their participation in the development of various conditions, both in health and pathology.

An Integrative Omics Approach Reveals Involvement of BRCA1 in Hepatic Metastatic Progression of Colorectal Cancer.

(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student’s t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting Brca1. We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.

Novel mutations in the RING-finger domain of BRCA1 gene in clinically diagnosed breast cancer patients.

In the present study, we investigated the frequency of BRCA1 gene mutations in 30 breast cancer (BC) patients of independent family history and 30 healthy control subjects. The immunohistochemistry (IHC) of BCpatients showed duct cell carcinoma and distinct expression of the human epidermal growth factor receptor2 (HER2). The genomic DNA was extracted from the BC patients and control subjects, the BRCA1 gene was PCR amplifiedand sequenced. The sequence analysis revealed that BRCA1 gene mutations were detected in 5/30 (16.6%) unrelated patients. One novel deleterious c.53delT mutation was detected in 3/30 (10%) unrelated patients leading to p.Met18Serfs*5 frame shift mutation in exon 2. Two patients 2/30 (6%) had novel c.297_301delinsCTCAA mutation in exon 5 leading to p.Leu99_Tyr101delinsPheSerAsn. Interestingly, the qRT-PCR analysis showed high expression of BRCA1 gene in all these patients having mutations compared with control subjects. Further, in silico analysis revealed loss of zinc-binding region ofthe RING-finger domain in BRCA1 structure due to these mutations, variable number of helices, helix-helix interactions, β-turns, and γ-turns were identified in the secondary structure, resulted in the formation of non-functional protein which is unable to activate BRCA1-associated genome surveillance complex (BASC) leading to uncontrolled cell proliferation. Moreover, the molecular dynamics (MD) simulations of mutated BRCA1 protein demonstrated extensive variations in the domain and non-domain regions compared with the wild-type structure as indicated by RMSD values. All these results conclusively explain that the c.53delT mutation may be the probable founder of deleterious mutation in this ethnic group.

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and 212Pb-trastuzumab (Pac/212Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/212Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/212Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that 212Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

Helicase-Like Transcription Factor (Hltf) Regulates G2/M Transition, Wt1/Gata4/Hif-1a Cardiac Transcription Networks, and Collagen Biogenesis

HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum) brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf’s regulation of the G2/M transition (p=9.726E-15) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2) and lysyl (Plod2) collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3) for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus, silencing Hltf during heart organogenesis compromised DNA double-strand break repair, and caused aberrant collagen biogenesis altering the structural network that transmits cardiomyocyte force into muscle contraction.

Abstract 1990: Effects of pomegranate extract on proliferation, apoptosis and mRNA expression in human breast cancer cells

Abstract Pomegranates (Punica granatum L.) contain a mixture of phytochemicals such as ellagitannins, ellagic acid and anthocyanins and possess antioxidant and anticancer activities. Pomegranate juice, aqueous pomegranate extract or seed oil reduces growth of different cancer cell lines and human prostate and lung cancer xenografts. Although several potential mechanisms such as stimulation of apoptosis, inhibition of inflammation and angiogenesis have been identified, further mechanistic investigations, especially in breast cancer, are needed. In this study, we examined the growth inhibitory effects of PE in association with antioxidant activity and global gene expression changes in MCF-7 breast cancer cells. PE caused a time and concentration-dependent inhibition of cell proliferation associated with the G2/M cell cycle arrest. In addition, PE induced apoptosis in a concentration-dependent manner. High antioxidant activity of PE was confirmed using the Trolox Equivalent Antioxidant Capacity assay (Trolox assay). Our data shows that 50 µg/ml of PE, which inhibits 50% of cell growth, is equivalent to 75 µg/ml of Trolox, a water-soluble vitamin E analog, and 40 µg/ml of N-acetyl Cysteine, a well-known low molecular weight thiol antioxidant. To understand whether the antioxidant activity of PE contributed to the observed cell growth inhibition, we determined whether PE (dose range 0-50 µg/ml) and equivalent doses of N-acetyl Cysteine and Trolox inhibit MCF-7 cell growth. Only PE but not equivalent doses N-acetyl Cysteine and Trolox inhibited cell growth suggesting that the growth inhibition of PE cannot solely be attributed to its antioxidant potential. To get insight into the molecular mechanisms of action of PE we conducted gene expression profiling on the cells treated with 50% growth inhibitory dose of PE using Affymetrix Human Gene 1.0 ST Array. Our data showed that 903 gene entities were altered by PE treatment (1.5-fold cut-off, p&amp;lt;0.05), from which 398 were downregulated, while 505 were upregulated. Most of the genes that were downregulated by PE treatment affect processes associated with mitosis, chromosomal organization and segregation, DNA replication, DNA recombination and repair, whereas most upregulated genes constitute genes involved in the regulation of apoptosis and cell cycle. Of particular interest, PE downregulated important DNA damage response and repair genes including genes of MRE11-RAD50-NBS1 complex and BRCA1-associated genome surveillance complex. Given that targeting of DNA repair in cancer may sensitize cancer cells to drugs, these findings may have a translational potential. We are currently validating the genes at their mRNA and protein levels to identify novel ‘pomegranate-target gene’ interactions that are relevant to breast cancer. It may provide novel molecular and cellular targets for pomegranate-based anticancer approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1990. doi:1538-7445.AM2012-1990

Predicting protein complexes by data integration of different types of interactions

The explosion of high throughput interaction data from proteomics studies gives us the opportunity to integrate Protein-Protein Interactions (PPI) from different type of interactions. These methods rely on the assumption that proteins within a complex have more interactions across the different data sets which translate into the identification of dense subgraphs. However, the relative importance of the types of interaction are not equivalent in their reliability and accuracy consequently they should be analysed separately. Here we propose a method that use graph theory and mathematical modelling to solve this problem. Our approach has four steps that: i) score independently each type of interaction; ii) build an interaction specific networks for each type; iii) weight the specific networks; and iv) combine and normalise the scores. Using this approach to the BRCA1 Associated genome Surveillance Complex (BASC), we correctly identified the known core components of the complex and subcomplexes that have solved structures as well as predicted new interactions and core complexes. The method presented in this study is of general use. It is flexible enough to allow the development of any scoring system and can be applied to any protein complex to provide the latest knowledge in its interactions and structure.

ATM Activation by Ionizing Radiation Requires BRCA1-associated BAAT1

ATM (ataxia telangiectasia mutated) is required for the early response to DNA-damaging agents such as ionizing radiation (IR) that induce DNA double-strand breaks. Cells deficient in ATM are extremely sensitive to IR. It has been shown that IR induces immediate phosphorylation of ATM at Ser(1981), leading to catalytic activation of the protein. We recently isolated a novel BRCA1-associated protein, BAAT1 (BRCA1-associated protein required for ATM activation-1), by yeast two-hybrid screening and found that BAAT1 also binds to ATM, localizes to double-strand breaks, and is required for Ser(1981) phosphorylation of ATM. Small interfering RNA-mediated stable or transient reduction of BAAT1 resulted in decreased phosphorylation of both ATM at Ser(1981) and CHK2 at Thr(68). Treatment of BAAT1-depleted cells with okadaic acid greatly restored phosphorylation of ATM at Ser(1981), suggesting that BAAT1 is involved in the regulation of ATM phosphatase. Protein phosphatase 2A-mediated dephosphorylation of ATM was partially blocked by purified BAAT1 in vitro. Significantly, acute loss of BAAT1 resulted in increased p53, leading to apoptosis. These results demonstrate that DNA damage-induced ATM activation requires a coordinated assembly of BRCA1, BAAT1, and ATM.

The absence of a functional relationship between ATM and BLM, the components of BASC, in DT40 cells.

Bloom syndrome (BS) and ataxia-telangiectasia (A-T) are rare autosomal recessive diseases associated with chromosomal instability. The genes responsible for BS and A-T have been identified as BLM and ATM, respectively, whose products were recently found to be components of BRCA1-associated genome surveillance complex (BASC), a supercomplex possibly involved in the recognition and repair of aberrant DNA structures. Based on experiments using BLM(-/-) DT40 cells and BLM(-/-)/RAD54(-/-) DT40 cells, we previously suggested that BLM functions to reduce the formation of double-strand breaks (DSBs) during DNA replication. To examine whether ATM is involved in the recognition and/or repair of DSBs generated in BLM(-/-) DT40 cells and to address the functional relationship between the two BASC components, we generated BLM(-/-)/ATM(-/-) DT40 cells and characterized their properties as well as those of ATM(-/-) and BLM(-/-) DT40 cells. BLM(-/-)/ATM(-/-) cells proliferated slightly more slowly than either BLM(-/-) or ATM(-/-) cells. The sensitivity of BLM(-/-)/ATM(-/-) cells to gamma-irradiation was similar to that of ATM(-/-) cells, while BLM(-/-) cells were slightly resistant to gamma-irradiation compared with wild-type cells. BLM(-/-) cells showed sensitivity to methyl methanesulfonate (MMS) and UV irradiation while ATM(-/-) cells did not show sensitivity to either agent. The sensitivity of BLM(-/-)/ATM(-/-) cells to MMS and UV was similar to that of BLM(-/-) cells. Disrupting the function of ATM reduced the targeted integration frequency in BLM(-/-) DT40 cells. However, a defect in ATM only slightly reduced the increased sister chromatid exchanges (SCEs) in BLM(-/-) DT40 cells.

Mismatch repair: praying for genome stability

Over the past years additional functions have emerged for mismatch repair proteins. The first was the involvement of mismatch repair proteins with homologous DNA recombination, one of the pathways that repair DNA double-strand breaks. A key intermediate in this repair process is heteroduplex joint formation between the ends of the broken molecule and an intact, homologous partner (Fig. 3Fig. 3). During meiosis, the specialized MutS-homologous dimer MSH4–MSH5 stimulates the induction of crossovers at joints between homologous autosomes, in conjunction with the MutL homologues (Fig. 3Fig. 3a). Polymorphisms, commonly present in homologous autosomes, are apparent as mismatches within the joints. MSH2–MSH6, together with the MutL homologues, corrects these mismatches in a process called gene conversion (Fig. 3Fig. 3a,b). Double strand breaks in somatic cells, such as those induced by X-rays, may result in translocations or in loss of genetic information when homologous recombination takes place between nonidentical sequences, such as pseudogenes or homologous autosomes, rather than between identical sister chromatids. In these cells, mismatch repair generally does not perform gene conversion, but instead antagonizes mismatch-containing heteroduplex joints in a process called anti-recombination (Fig. 3Fig. 3c). Absence of mismatch repair, therefore, allows crossover between diverged sequences. As an example, crosses between E. coli and mismatch-repair-deficient Salmonella typhimurium cells result in interspecies hybrids, because crossovers between the 20% diverged genomes now proceed unrestrained.Fig. 3Roles of mismatch repair in determining the outcome of homologous DNA recombination. Top: both ends of each strand of a broken, gapped, DNA molecule (gray backbone) form a heteroduplex junction with a homologous DNA molecule (orange backbone), each consisting of two heteroduplex joints. Note that this represents a simplification of the Holliday model. Sequence divergences between the recombining molecules are apparent as mismatches (M). Mismatch repair proteins can determine the outcome of recombination in three ways: (a) crossover, resulting in an exchange of flanking DNA. This generally is accompanied by repair of the mismatch (‘gene conversion’, indicated by CM). (b) Gene conversion without crossover. (c) Anti-recombination by mismatch repair proteins in somatic cells results in reversion of mismatch-containing heteroduplexes.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn mammalian cells, mismatch repair is involved in triggering apoptosis induced by certain DNA damaging agents. The best examples are agents that methylate the O6 position of guanosine residues, including certain carcinostatic drugs. Wild-type cells are extremely sensitive to these drugs, as incorporation of either C or T opposite O6Me–G residues triggers mismatch-repair-dependent excision. However, resynthesis introduces the same ‘mismatch’, resulting in futile cycles of mismatch repair that ultimately result in apoptosis. An alternative explanation for the mismatch repair dependence of the toxicity of these agents is provided by the similarity of MSH2–MSH6 to Ras nucleotide exchange factors that are active in a GTP-bound form and inactive in a GDP-bound form. MSH2–MSH6, binding to the methylated mispair, exchanges ATP for ADP and might directly signal to downstream effectors of apoptosis. Supporting this model is the detection of mismatch repair proteins in the so-called BRCA1-associated genome surveillance complex, implicated in signaling of DNA damage to cell cycle regulators.Mismatch repair proteins are implicated in several other DNA repair processes, including repair of oxidative nucleotide damage in the transcribed strand of genes, in repair of DNA crosslinks, and in the processing of DNA ends during the single-strand annealing pathway of double strand break repair. All these seemingly diverse activities justify the assignment to mismatch repair of the honorary title ‘Caretaker of our Genome’.

Damage Report

The DNA damage checkpoint was originally described as a regulatory mechanism that was dispensable in the absence of damage, but caused arrest of the cell cycle once DNA damage was detected. In a review, Zhou and Elledge provide a more expansive view of the cellular response to DNA damage and suggest that the "checkpoint" itself is best understood as part of a larger process (which they term the "DNA damage response") that influences not only control of cell-cycle progression, but also of DNA repair mechanisms, telomere length, apoptosis, and other processes. They discuss proposed sensors of DNA damage, which include BASC (the BRCA1-associated genome surveillance complex), a large complex that comprises the breast cancer-associated tumor suppressor gene product BRCA1, the ataxia telangiectasia gene product ATM, and the Nijmegen breakage syndrome protein (Nbs1). A summary of the better-understood transducers and effectors of the damage response is followed by a look at some of the fascinating questions that remain to be resolved. For example, it is not enough to sense DNA damage. The cell also must sense the completion of the repair process. We have a sense of how E. coli manages this, but in mammalian systems, the primary subject of this review, a solution has yet to be described. Zhou, B.-B.S., and Elledge, S.J. (2000) The DNA damage response: Putting checkpoints in perspective. Nature 408 : 433-439. [Online Journal]

BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures

We report the identities of the members of a group of proteins that associate with BRCA1 to form a large complex that we have named BASC (BRCA1-associated genomesurveillance complex). This complex includes tumor suppressors and DNA damage repair proteins MSH2, MSH6, MLH1, ATM, BLM, and the RAD50–MRE11–NBS1 protein complex. In addition, DNA replication factor C (RFC), a protein complex that facilitates the loading of PCNA onto DNA, is also part of BASC. We find that BRCA1, the BLM helicase, and the RAD50–MRE11–NBS1 complex colocalize to large nuclear foci that contain PCNA when cells are treated with agents that interfere with DNA synthesis. The association of BRCA1 with MSH2 and MSH6, which are required for transcription-coupled repair, provides a possible explanation for the role of BRCA1 in this pathway. Strikingly, all members of this complex have roles in recognition of abnormal DNA structures or damaged DNA, suggesting that BASC may serve as a sensor for DNA damage. Several of these proteins also have roles in DNA replication-associated repair. Collectively, these results suggest that BRCA1 may function as a coordinator of multiple activities required for maintenance of genomic integrity during the process of DNA replication and point to a central role for BRCA1 in DNA repair.