BRCT Repeats Research Articles

Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5'-terminated strands at DSB sites1,2. The breast cancer suppressor BRCA1-BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress1,3-9. BRCA1-BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear10-16. Using purified recombinant proteins, we show here that BRCA1-BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11-RAD50-NBS1 and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex17,18, which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1-C complex19-21, inhibits resection, showing that BRCA1-C is a functionally integrated ensemble. Whereas BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4-6,8). We show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1-BARD1 in various physiological contexts.

Aminoguanidinium hydrogen 1,1-cyclobutane dicarboxylate: Synthesis, structural characterization and molecular docking studies

On neutralization of Aminoguanidine bicarbonate (H2Agun) with 1,1 -cyclobutane dicarboxylic acid (H2CBD) resulted aminoguanidinium salt, [(HAgun)+(HCBD)-]. Using FT-IR, 1H NMR, 13C NMR, and analytical spectroscopy, the compound was identified and characterized. Furthermore, single X-ray diffraction experiment confirms the molecular structure of the salt. The molecular docking approach has been employed to investigate the inhibitory properties of aminoguanidinium hydrogen 1,1-cyclobutane dicarboxylate on three key cancer protein receptors: 3WZE (VEGFR kinase), 5ZMA (crystal structure of an allosteric Eya2 phosphates inhibitor lung cancer protein), and 1JNX (BRCT repeat region from the breast cancer associated protein). Observation of lower binding energy of −4.67 kcal/mol for 3WZE, −4.57 kcal/mol for 5ZMA and 1.7 kcal/mol for 1JNX, respectively, indicating high stability with the protein molecule.

Polygonum aviculare L.'s biological Activities: Investigating its Anti-Proliferative, Antioxidant, chemical properties supported by molecular docking study

In this study, two different solvent extracts of Polygonum aviculare L. collected from natural environment were evaluated in terms of their antioxidant properties, antiproliferative properties and interactions of the extract components with proteins, and were addressed in terms of their pharmacological potential and biological activities. The plant was extracted using two different solvents, and its biological activity levels were examined utilizing in vitro antioxidant and antiproliferative (cytotoxicity assay) assays. According to the results obtained, there was a variation in chemical composition between the water and methanol extracts of this plant. While “1,4:3,6-Dianhydro-.alpha.-d-glucopyranose” was determined with 2.78 as the major component of the water extract of the plant, “n-Hexadecanoic acid” was determined with 10.32 in the methanol extract. The antioxidant activity of the plant was evaluated according to both DPPH and ABTS methods. The results obtained in both methods were found to be close to each other. While the antioxidant activity value of the plant remained at a moderate level compared to the standard substance, it was observed that the activity value increased as the concentration increased. When the results were examined in terms of antiproliferative properties of aqueous and methanolic extracts, it was seen that only the water extract was active. In addition, among the molecules identified in the extracts, it was determined by molecular docking calculations that cyclododecane, which is the third most abundant component in the methanol extract, showed the highest biological activity. In the same time, the activities of chemicals found in Polygonum aviculare L. plant extract against a range of proteins, including the crystal structures of the following: bovine xanthine oxidedase (PDB ID: 3NRZ), human peroxiredoxin 5 (PDB ID: 1HD2), antibacterial FabH inhibitors (PDB ID: 4Z8D), estrogen receptor (PDB ID: 1A52), and the BRCT repeat region (PDB ID: 1JNX). Next, the compounds with the highest activity were analyzed for their ADME/T characteristics.

Structural insights into Rad18 targeting by the SLF1 BRCT domains

Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18 recognition by SLF1 is unclear. Here, we present the crystal structure of the tandem BRCT repeat (tBRCT) in SLF1 (SLF1tBRCT) bound with the interacting Rad18 peptide. Our structure and biochemical studies demonstrate that SLF1tBRCT interacts with two phosphoserines and adjacent residues in Rad18 for high-affinity and specificity Rad18 recognition. We found that SLF1tBRCT utilizes mechanisms common among tBRCTs as well as unique ones for Rad18 binding, the latter include interactions with an α-helical structure in Rad18 that has not been observed in other tBRCT-bound ligand proteins. Our work provides structural insights into Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated complex assembly.

Synthesis of novel Cu(II) complexes with N2O2 ligands: Characterization, theoretical calculations, antimicrobial, antioxidant, DNA binding, and in vitro anticancer activity studies

The aim was to investigate the in vitro antimicrobial, antioxidant, cytotoxic activities and pBR322 Plasmid DNA interaction potentials of newly synthesized Schiff bases and their copper complexes. Cytotoxic activities of these compounds were determined by MTT methods in human breast cancer (MCF-7), human cervical cancer (HeLa) and mouse fibroblast (L929) cell lines. The determination of changes in oxidant and antioxidant load of the cell after application of these compounds were investigated with the aid of Rel Assay Diagnostics kits. It was found that the some of the compounds had moderate antimicrobial activity, and oxidative stress index. According to plasmid DNA interaction studies, synthesized complexes modified the tertiary structure of pBR322 plasmid DNA. The compound Bis-Napht showed the highest cytotoxic activity in HeLa, MCF-7 and L929 cells at an IC50 dose of 3 ± 1 µM, 5 ± 1 µM and 3 ± 1 µM respectively. Compound Bis-Sal was determined to have lower cytotoxic activity in L-929 cells at an IC50 dose of 106 ± 9 µM at 24 h compared to other compounds. Ligand compounds and their metal complexes were optimized on the B3LYP, HF, and M06-2x methods with the 6–31++g(d,p) basis set. Afterwards, their activities were compared against crystal structure of the BRCT repeat region from the breast cancer associated protein, (BRCA1) (PDB ID: 1JNX) and crystal Structure of CDK2 receptor of cervical cancer cell proteins (PDB ID: 4BGH). To examine the interactions occurring in more detail, Protein-Ligand Interaction Profiler analysis was performed and all chemical interactions that occurred were determined.

In Silico and Structure-Based Assessment of Similar Variants Discovered in Tandem Repeats of BRCT Domains of BRCA1 and BARD1 To Characterize the Folding Pattern.

BRCA1 and BARD1 are important proteins in the homologous DNA damage repair pathways. Different genetic variants identified in these proteins have been clinically correlated with the occurrence of hereditary breast and ovarian cancer (HBOC). Variants of unknown significance (VUS) reported in the BRCT domains of BRCA1 and BARD1 substantiate the importance of BRCT domain-containing proteins for genomic integrity. To classify the pathogenicity of variants, in silico, structural and molecular dynamics (MD)-based approaches were explored. Different variants reported in the BRCT region were retrieved from cBioPortal, LOVD3, BRCA Exchange, and COSMIC databases to evaluate the pathogenicity. Multiple sequence alignment and superimposition of the structures of BRCA1 BRCT and BARD1 BRCT domains were performed to compare alterations in folding patterns. From 11 in silico predictions servers, variants reported to be pathogenic by 70% of the servers were considered for structural analysis. To our observations, four residue pairs of both the proteins were reported, harboring 11 variants, H1686Y, W1718L, P1749L, P1749S, and W1837L variants for BRCA1 BRCT and H606D, H606N, W635L, P657L, P657S, and W762F for BARD1 BRCT. MD simulations of the BRCT repeat regions of these variants and wild-type proteins were performed to evaluate the differences of folding patterns. Root mean square deviation (RMSD), R g, solvent-accessible surface area (SASA), and root mean square fluctuation (RMSF) of variants showed slight differences in the folding patterns from the wild-type proteins. Furthermore, principal components analysis of H1686Y, P1749S, and W1718L variants of BRCA1 showed less flexibility than the wild type, whereas that of H606D, W635L, and W762F of BARD1 showed more flexibility than the wild type. Normal mode analysis of the energy minima from the simulation trajectories revealed that most of the variants do not show much differences in the flexibility compared to the wild-type proteins, except for the discrete regions in the BRCT repeats, most prominently in the 1798-1801 amino acid region of BRCA1 and at the residue 744 in BARD1.

Anti-breast Adenocarcinoma and Anti-urease Anti-tyrosinase Properties of 5-Pentylresorcinol as Natural Compound with Molecular Docking Studies.

5-Pentylresorcinol is a type of the group of resorcinol compounds that is resorcinol in that has hydrogen atom at position 5 is replaced by a pentyl group. It has a role as a lichen metabolite. This compound showed excellent to good inhibitory activities against studied these enzymes with IC50 values of 65.96 µM for urease and 34.81 µM for tyrosinase. Standard compounds for enzymes had IC50 values of 1.94±0.24 µM against urease and 84.36±5.17 µM against tyrosinase. The IC50 of 5-pentylresorcinol against MCF7 cell line was 165.72 µg/mL; against Hs 578Bst cell line was 102.14 µg/mL; against Hs 319.T cell line was 12.34 µg/mL; and against UACC-3133 cell line was 73.07 µg/mL, respectively. The chemical activities of 5-pentylresorcinol against urease and tyrosinase were evaluated using the molecular modeling study. The anti-cancer activity of 5-pentylresorcinol was also investigated by treating the compound on the BRCT repeat region from the breast cancer-associated protein (BRCA1), and their interactions were assessed utilizing the molecular docking calculations. The results revealed the probable interactions and their characteristics at an atomic level. The docking scores of 5-pentylresorcinol against urease, tyrosinase, and BRCA1 are -3.073, -5.262, and -3.238 (kcal/mol), respectively.

Synthesis, bioinformatics and biological evaluation of novel pyridine based on 8-hydroxyquinoline derivatives as antibacterial agents: DFT, molecular docking and ADME/T studies

• Synthesis of new compounds based on 8-hydroxyquinoline and their characterization by FT-IR, 1 H NMR, 13 C NMR and elemental analyses. • The antibacterial activity of all the compounds was carried out for five Gram-positive and Gram-negative strains. • The minimum inhibitory concentrations (MICs) of active compounds were calculated and discussed. • The chemical and biological activities of 8-Hydroxyquinoline derivatives were compared with theoretical methods (DFT, molecular docking, ADME/T). The present study aims to first explore the relationship between the chemical structure of the organic compounds (pyridine based on 8-hydroxyquinoline) and the antibacterial activities, as well as the impact of substituent effects on their antibacterial properties. These compounds were synthesized by efficient methods, and their structures confirmed by the spectral methods (IR, 1 H NMR, 13 C NMR and elemental analysis). The antibacterial activities of synthesized the compounds were checked with five Gram-positive and Gram-negative strains such as Enterobacter cloacae (ATCC29893),Escherichia coli (ATCC35218), Klebsiella pneumoniae (ATCC13883), Staphylococcus aureus ( ATCC29213 ) and Acinetobacter baumannii (ATCC19606) . The results of the antibacterial activities of four synthesized compounds were compared with three standard antibiotics [ Penicillin G, Erythromycin and Norfloxacin ( quinoline type)]. The minimum inhibitory concentrations (MICs) of active compounds were calculated and discussed. The chemical and biological activities of hydroxyquinoline derivatives were compared with theoretical methods. The chemical activities of hydroxyquinoline derivatives were contrasted with the important quantum chemical parameters using the HF/6–31++ g ( d, p ) basis sets. Besides, biological activities of hydroxyquinoline derivatives against cancer proteins that are respectively protein of the BRCT repeat region of breast cancer that is ID: 1JNX, crystal structure of liver cancer protein that is ID: 3WZE, and crystal structure of lung cancer protein that is ID: 5ZMA, were compared. ADME/T (Adsorption, Distribution, Metabolism, Excretion, and Toxicity) analysis was studied for molecules with high biological activity to become efficient drugs in the future.

Structurally diverse heterobimetallic Pb(II)-Salen complexes mechanistic notion of cytotoxic activity against neuroblastoma cancer cell: Synthesis, characterization, protein–ligand interaction profiler, and intuitions from DFT

In this work, two new heterobimetallic Pb(II) complexes,namely [Zn(L1)(η1-NCS)Pb(η1-SCN)] (1) and [Cd(L2)(η1-NCS)Pb(η1-SCN)]n(2) integrated from Salen ligands (L1/L2) in the presence of NaSCN. The title compounds were characterized with microanalytical, spectroscopic techniques, and SC-XRD. SC-XRD divulges a de-protonated form of ligands trapped by M(II) ions into the N2O2 compartment (M = Zn/Cd), whereas more compelling Pb(II) ions outer O4 cavities. The tethering thiocyanate linkers formed the discrete (1) and the 1D polymer (2). We accomplished the DFT using B3LYP, HF, and M062X with Lanl2dz level basis sets to delineate Frontier molecular orbital (FMO), the complex biological activity, and the Global chemical parameters. Molecular docking (MD) finds the plausible binding mechanism by implanting the complexes into the active site of crystal systems of the BRCT repeat region from the breast cancer-associated protein, VEGFR kinase liver cancer protein, and an allosteric Eya2 lung cancer protein. Polar and hydrophobic exchanges, π-π interaction, hydrogen bonds, and Bromine are the essential binding process. Protein-Ligand Interaction Profiler (PLIP) examined the interaction of the protein with complex 1.We consider the mechanistic perception of cytotoxic action, apoptosis cancer cell death via intracellular reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) of apoptotic cells withSH-SY5Y human neuroblastoma cancer cells.The enhanced ROS generation and mitochondrial depolarization in SH-SY5Y cells indicate apoptosis in these cells. We explain the Western blot-activated caspase-3 in lysates of SH-SY5Y cells for the apoptotic pathway, exploring the induction of apoptotic pathwaysvia1and2in neuroblastoma cells.

Investigation of structural, electronical and in vitro cytotoxic activity properties of some heterocyclic compounds

A series of heterocyclic compounds (15) were synthesized, characterized and tested towards two human cancer cell lines for learning their in vitro antiproliferative activities. Compound 3 demonstrated the most promising activity in breast cancer cell line with a half maximal inhibitory concentration (IC50) value of 23.73 μM compared to other compounds (1, 2, 4, 5). Cytotoxic activity studies revealed that compounds 24 did not have antiproliferative activity towards liver cancer cell line. Computational methods were used to determine various quantum chemical parameters in order to identify correlations with the measured biological activity, which can assist in the molecular modeling of new heterocyclic systems. The biological activities of heterocyclic molecules against cancer cell proteins that are the crystal structure of the BRCT repeat region from the breast cancer-associated protein, ID: 1JNX, crystal structure of VEGFR kinase (liver cancer) protein, ID: 3WZE, and crystal structure of an allosteric Eya2 phosphatase inhibitor (lung cancer) protein, ID: 5ZMA, were compared. Finally, ADME/T analysis was performed for heterocyclic molecules and their future possibilities as a drug were investigated.

The repair gene BACH1 - a potential oncogene.

BACH1 encodes for a protein that belongs to RecQ DEAH helicase family and interacts with the BRCT repeats of BRCA1. The N-terminus of BACH1 functions in DNA metabolism as DNA-dependent ATPase and helicase. The C-terminus consists of BRCT domain, which interacts with BRCA1 and this interaction is one of the major regulator of BACH1 function. BACH1 plays important roles both in phosphorylated as well as dephosphorylated state and functions in coordination with multiple signaling molecules. The active helicase property of BACH1 is maintained by its dephosphorylated state. Imbalance between these two states enhances the development and progression of the diseased condition. Currently BACH1 is known as a tumor suppressor gene based on the presence of its clinically relevant mutations in different cancers. Through this review we have justified it to be named as an oncogene. In this review, we have explained the mechanism of how BACH1 in collaboration with BRCA1 or independently regulates various pathways like cell cycle progression, DNA replication during both normal and stressed situation, recombination and repair of damaged DNA, chromatin remodeling and epigenetic modifications. Mutation and overexpression of BACH1 are significantly found in different cancer types. This review enlists the molecular players which interact with BACH1 to regulate DNA metabolic functions, thereby revealing its potential for cancer therapeutics. We have identified the most mutated functional domain of BACH1, the hot spot for tumorigenesis, justifying it as a target molecule in different cancer types for therapeutics. BACH1 has high potentials of transforming a normal cell into a tumor cell if compromised under certain circumstances. Thus, through this review, we justify BACH1 as an oncogene along with the existing role of being a tumor suppressant.

Efficient synthesis and molecular docking studies of new pyrimidine-chromeno hybrid derivatives as potential antiproliferative agents

Various novel heterocyclic compounds containing pyrimidine nuclei 5H-chromeno[4,3-d]pyrimidine (4a–c, e–h, l–r, t) and pyrimidine-5-yl-(2-hydroxyphenyl)methanone (5a, c, d, f–k, m–o, r, s, u) were synthesized from the reaction of guanylhydrazones (2a–u) and 3-formylchromone (3). These compounds were tested against human liver hepatocellular carcinoma cell line (HepG2) and human breast adenocarcinoma cell line (MDA-MB-231) using the MTT assay method. Furthermore, molecular docking calculations were performed to compare the biological activities of various novel heterocyclic compounds against cancer proteins. In these calculations, the protein used are crystal structure of the BRCT repeat region from the breast cancer associated protein, 1JNX, crystal structure of VEGFR kinase (liver cancer) protein, 3WZE, and crystal structure of an allosteric Eya2 phosphates inhibitor (lung cancer) protein, 5ZMA, respectively. After molecular docking calculations, absorption, distribution, metabolism, and excretion/toxicity analysis was performed to examine the properties of various novel heterocyclic compounds for their future use as drugs.

Anti-quorum sensing activity in Pseudomonas aeruginosa PA01 of benzimidazolium salts: electronic, spectral and structural investigations as theoretical approach

Quorum sensing (QS) is a system used in the expression of virulence factors by many pathogenic bacteria, and blockage of the system is seen as a new and effective strategy in combating with resistant bacteria. The inhibition effects of two benzimidazolium salts, namely 1-(2-methylbenzonitrile)-3-benzylbenzimidazolium bromide (2) and 1-(N-methylphthalimide)-3-(4-methylbenzyl)benzimidazolium bromide (3), on quorum sensing-related virulence factors such as pyocyanin, elastase, biofilm formation and swarming motility, which is an opportunistic pathogen Pseudomonas aeruginosa PA01, were investigated in this study. The results show that the compound 3 has a significant inhibition on biofilm formation with 94%. Furthermore, the compounds 2 and 3 reduced swarming motility by 64–69% as well as pyocyanin production by 49–64% in P. aeruginosa PA01 without preventing bacterial growth in the tested concentrations. HF, B3LYP and M06–2X methods were used with 3–21 g, 6–31 g, sdd basis sets to compare the chemical activity of the compounds. Theoretically, 1H NMR, 13C NMR and Infrared spectra of the compounds were calculated in the HF/6-31++g basis set. The biological activities of the relative compounds were theoretically studied against cancer proteins. Crystal structure of the BRCT repeat region from the breast cancer associated protein, ID: 1JNX, crystal structure of liver cancer protein, ID: 3WZE and crystal structure of lung cancer protein, ID: 5ZMA, were compared. In the docking studies, the best result was obtained with compound 2 against the lung cancer cell with a docking score parameter of −5.85. Communicated by Ramaswamy H. Sarma

Structure-function analysis of TOPBP1\u2019s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system

The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1’s nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.

A Non-canonical BRCT-Phosphopeptide Recognition Mechanism Underlies RhoA Activation in Cytokinesis

Cytokinesis partitions the cell contents to complete mitosis. During cytokinesis, polo-like kinase 1 (PLK1) activates the small GTPase RhoA to assemble a contractile actomyosin ring. PLK1 is proposed to pattern RhoA activation by creating a docking site on the central spindle that concentrates the RhoA guanine nucleotide exchange factor ECT2. However, ECT2 targeting to the central spindle is dispensable for cytokinesis, indicating that how PLK1 controls RhoA activation remains unresolved. To address this question, we employed an unbiased approach targeting ∼100 predicted PLK1 sites in two RhoA regulators: ECT2 and the centralspindlin complex, composed of CYK4 and kinesin-6. This comprehensive approach suggested that the only functionally critical PLK1 target sites are in a single cluster in the CYK4N terminus. Phosphorylation of this cluster promoted direct interaction of CYK4 with the BRCT repeat module of ECT2. However, mutational analysis invitro and invivo led to the surprising finding that the interaction was independent of the conserved "canonical" residues in ECT2's BRCT repeat module that, based on structurally characterized BRCT-phosphopeptide interactions, were presumed critical for binding. Instead, we show that the ECT2 BRCT module binds phosphorylated CYK4 via a distinct conserved basic surface. Basic surface mutations mimic the effects on cytokinesis of loss of CYK4 cluster phosphorylation or inhibition of PLK1 activity. Together with evidence for ECT2 autoinhibition limiting interaction with CYK4 in the cytoplasm, these results suggest that a spatial gradient of phosphorylated CYK4 around the central spindle patterns RhoA activation by interacting with ECT2 on the adjacent plasma membrane.

Classification of VUS and unclassified variants in BRCA1 BRCT repeats by molecular dynamics simulation

Pathogenic mutation in BRCA1 gene is one of the most penetrant genetic predispositions towards cancer. Identification of the mutation provides important aspect in prevention and treatment of the mutation-caused cancer. Of the large quantity of genetic variants identified in human BRCA1, substantial portion is classified as Variant of Uncertain Significance (VUS) or unclassified variants due to the lack of functional evidence. In this study, we focused on the VUS and unclassified variants in BRCT repeat located at BRCA1 C-terminal. Utilizing the well-determined structure of BRCT repeats, we measured the influence of the variants on the structural conformations of BRCT repeats by using molecular dynamics simulation (MDS) consisting of RMSD (Root-mean-square-deviation), RMSF (Root-mean-square-fluctuations), Rg (Radius of gyration), SASA (Solvent accessible surface area), NH bond (hydrogen bond) and Covariance analysis. Using this approach, we analyzed 131 variants consisting of 89 VUS (Variant of Uncertain Significance) and 42 unclassified variants (unclassifiable by current methods) within BRCT repeats and were able to differentiate them into 78 Deleterious and 53 Tolerated variants. Comparing the results made by the saturation genome editing assay, multiple experimental assays, and BRCA1 reference databases shows that our approach provides high specificity, sensitivity and robust. Our study opens an avenue to classify VUS and unclassified variants in many cancer predisposition genes with known protein structure.

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.

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.

Structural and functional characterization of the PNKP-XRCC4-LigIV DNA repair complex.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4–LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP–XRCC4–LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4–LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4–LigIV that regulate PNKP recruitment and activity within NHEJ.

Mdc1 modulates the interaction between TopBP1 and the MRN complex during DNA damage checkpoint responses

TopBP1 has been identified as a direct activator of ATR and interacts with the Nbs1 subunit of the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. In this study, we show that Mdc1 associates with both TopBP1 and Nbs1 in egg extracts and human cells. We cloned a cDNA encoding the full-length version of Xenopus Mdc1. The association between Mdc1 and TopBP1 involves the first pair of BRCT repeats in TopBP1. The N-terminal region (161–230) of Mdc1 is required for this binding. The interaction between Mdc1 and Nbs1 involves the two tandem BRCT repeats of Nbs1. Functional studies with mutated forms of Mdc1, TopBP1, and Nbs1 indicate that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. TopBP1 cannot interact with Nbs1 in Mdc1-depleted egg extracts, suggesting that Mdc1 connects TopBP1 and Nbs1 together. These findings suggest that Mdc1 is a crucial mediator of the DNA damage checkpoint response.