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1.
Elife ; 102021 07 13.
Article in English | MEDLINE | ID: mdl-34254584

ABSTRACT

Breast cancer type two susceptibility protein (BRCA2) is an essential protein in genome maintenance, homologous recombination (HR), and replication fork protection. Its function includes multiple interaction partners and requires timely localization to relevant sites in the nucleus. We investigated the importance of the highly conserved DNA-binding domain (DBD) and C-terminal domain (CTD) of BRCA2. We generated BRCA2 variants missing one or both domains in mouse embryonic stem (ES) cells and defined their contribution in HR function and dynamic localization in the nucleus, by single-particle tracking of BRCA2 mobility. Changes in molecular architecture of BRCA2 induced by binding partners of purified BRCA2 were determined by scanning force microscopy. BRCA2 mobility and DNA-damage-induced increase in the immobile fraction were largely unaffected by C-terminal deletions. The purified proteins missing CTD and/or DBD were defective in architectural changes correlating with reduced HR function in cells. These results emphasize BRCA2 activity at sites of damage beyond promoting RAD51 delivery.


Subject(s)
BRCA2 Protein/chemistry , BRCA2 Protein/genetics , DNA Repair , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Nucleic Acid Conformation , Animals , BRCA2 Protein/metabolism , DNA/chemistry , DNA/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Homologous Recombination , Humans , Mice , Mouse Embryonic Stem Cells , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Single Molecule Imaging
2.
Nucleic Acids Res ; 48(17): 9649-9659, 2020 09 25.
Article in English | MEDLINE | ID: mdl-32785644

ABSTRACT

BRCA2 is a key breast cancer associated protein that is predicted to have interspersed regions of intrinsic disorder. Intrinsic disorder coupled with large size likely allows BRCA2 to sample a broad range of conformational space. We expect that the resulting dynamic arrangements of BRCA2 domains are a functionally important aspect of its role in homologous recombination DNA repair. To determine the architectural organization and the associated conformational landscape of BRCA2, we used scanning force microscopy based single molecule analyses to map the flexible regions of the protein and characterize which regions influence oligomerization. We show that the N- and the C-terminal regions are the main flexible regions. Both of these regions also influence BRCA2 oligomerization and interaction with RAD51. In the central Brc repeat region, Brc 1-4 and Brc 5-8 contribute synergistically to BRCA2 interaction with RAD51. We also analysed several single amino acid changes that are potentially clinically relevant and found one, the variant of F1524V, which disrupts key interactions and alters the conformational landscape of the protein. We describe the overall conformation spectrum of BRCA2, which suggests that dynamic structural transitions are key features of its biological function, maintaining genomic stability.


Subject(s)
BRCA2 Protein/chemistry , BRCA2 Protein/metabolism , Rad51 Recombinase/metabolism , BRCA2 Protein/genetics , Humans , Microscopy, Atomic Force , Mutation, Missense , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , Rad51 Recombinase/genetics
3.
Nucleic Acids Res ; 45(8): 4507-4518, 2017 05 05.
Article in English | MEDLINE | ID: mdl-28168276

ABSTRACT

The tumor suppressor BRCA2 is a large multifunctional protein mutated in 50-60% of familial breast cancers. BRCA2 interacts with many partners and includes multiple regions with potentially disordered structure. In homology directed DNA repair BRCA2 delivers RAD51 to DNA resulting in removal of RPA and assembly of a RAD51 nucleoprotein filament. Dynamic rearrangements of BRCA2 likely drive this molecular hand-off initiating DNA strand exchange. We show human BRCA2 forms oligomers which can have an extended shape. Scanning force microscopy and quantitative single molecule fluorescence define the variety of BRCA2 complexes, reveal dramatic rearrangements upon RAD51 binding and the loading of RAD51 patches on single strand DNA. At sites of repair in cell nuclei, super-resolution microscopy shows BRCA2 and RAD51 arranged in largely separate locations. We identified dynamic structural transitions in BRCA2 complexes suggested to facilitate loading of RAD51 onto RPA coated single strand DNA and subsequent release of BRCA2.


Subject(s)
BRCA2 Protein/genetics , Cell Nucleus/genetics , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , Recombinational DNA Repair , Replication Protein A/genetics , BRCA2 Protein/chemistry , BRCA2 Protein/metabolism , Binding Sites , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , DNA Breaks, Single-Stranded , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Gene Expression , Humans , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/metabolism , Microscopy, Atomic Force , Protein Binding , Protein Multimerization , Replication Protein A/chemistry , Replication Protein A/metabolism , Single Molecule Imaging
5.
BMC Vet Res ; 11: 203, 2015 Aug 14.
Article in English | MEDLINE | ID: mdl-26268467

ABSTRACT

BACKGROUND: Elephants are classified as critically endangered animals by the International Union for Conservation of Species (IUCN). Elephant endotheliotropic herpesvirus (EEHV) poses a large threat to breeding programs of captive Asian elephants by causing fatal haemorrhagic disease. EEHV infection is detected by PCR in samples from both clinically ill and asymptomatic elephants with an active infection, whereas latent carriers can be distinguished exclusively via serological assays. To date, identification of latent carriers has been challenging, since there are no serological assays capable of detecting seropositive elephants. RESULTS: Here we describe a novel ELISA that specifically detects EEHV antibodies circulating in Asian elephant plasma/serum. Approximately 80 % of PCR positive elephants display EEHV-specific antibodies. Monitoring three Asian elephant herds from European zoos revealed that the serostatus of elephants within a herd varied from non-detectable to high titers. The antibody titers showed typical herpes-like rise-and-fall patterns in time which occur in all seropositive animals in the herd more or less simultaneously. CONCLUSIONS: This study shows that the developed ELISA is suitable to detect antibodies specific to EEHV. It allows study of EEHV seroprevalence in Asian elephants. Results confirm that EEHV prevalence among Asian elephants (whether captive-born or wild-caught) is high.


Subject(s)
Antibodies, Viral/blood , Elephants , Enzyme-Linked Immunosorbent Assay/veterinary , Herpesviridae Infections/veterinary , Herpesviridae/isolation & purification , Immunoglobulin G/isolation & purification , Animals , Animals, Zoo , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay/methods , Europe/epidemiology , Female , Herpesviridae/immunology , Herpesviridae Infections/diagnosis , Herpesviridae Infections/virology , Male , Sensitivity and Specificity , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism
6.
Nucleic Acids Res ; 35(17): 5819-30, 2007.
Article in English | MEDLINE | ID: mdl-17720710

ABSTRACT

Rad18 is a ubiquitin E3 ligase that monoubiquitinates PCNA on stalled replications forks. This allows recruitment of damage-tolerant polymerases for damage bypass and DNA repair. In this activity, the Rad18 protein has to interact with Rad6, the E2 ubiquitin-conjugating enzyme, ubiquitin, PCNA and DNA. Here we analyze the biochemical interactions of specific domains of the Rad18 protein. We found that the Rad6/Rad18 complex forms stable dimers in vitro. Consistent with previous findings, both the Ring domain and a C-terminal region contribute to the Rad6 interaction, while the C-terminus is not required for the interaction with PCNA. Surprisingly we find that the C2HC zinc finger is important for interaction with ubiquitin, apparently analogous to the interactions of classical zinc fingers with ubiquitin such as found in the UBZ and UBM domains in Y-family polymerases. Finally we find that the SAP domain, but not the zinc finger domain, is capable of DNA binding in vitro.


Subject(s)
DNA-Binding Proteins/chemistry , DNA/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin/metabolism , Animals , Binding Sites , DNA-Binding Proteins/metabolism , Dimerization , Mice , Protein Structure, Tertiary , Ubiquitin-Protein Ligases
7.
Nat Struct Mol Biol ; 12(7): 582-8, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15951818

ABSTRACT

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.


Subject(s)
Carrier Proteins/chemistry , Conotoxins/metabolism , Models, Molecular , Multiprotein Complexes/metabolism , Snails/chemistry , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Conotoxins/genetics , Conotoxins/pharmacology , Crystallography , Electrophysiology , Humans , Molecular Sequence Data , Mutation/genetics , Neurons/metabolism , Nicotinic Agonists/metabolism , Nicotinic Antagonists/metabolism , Oocytes/metabolism , Protein Binding , Protein Conformation , Sequence Alignment , Xenopus
8.
J Biol Chem ; 280(28): 26457-66, 2005 Jul 15.
Article in English | MEDLINE | ID: mdl-15899893

ABSTRACT

The crystal structure of acetylcholine-binding protein (AChBP) from the mollusk Lymnaea stagnalis is the established model for the ligand binding domains of the ligand-gated ion channel family, which includes nicotinic acetylcholine, 5-hydroxytryptamine (5-HT3), gamma-aminobutyric acid (GABA), types A and C, and glycine receptors. Here we present the crystal structure of a remote homolog, AChBP from Bulinus truncatus, which reveals both the conserved structural scaffold and the sites of variation in this receptor family. These include rigid body movements of loops that are close to the transmembrane interface in the receptors and changes in the intermonomer contacts, which alter the pentamer stability drastically. Structural, pharmacological and mutational analysis of both AChBPs shows how 3 amino acid changes in the binding site contribute to a 5-10-fold difference in affinity for nicotinic ligands. Comparison of these structures will be valuable for improving structure-function studies of ligand-gated ion channel receptors, including signal transduction, homology modeling, and drug design.


Subject(s)
Carrier Proteins/chemistry , Receptors, Cholinergic/chemistry , Receptors, Nicotinic/chemistry , Amino Acid Sequence , Animals , Binding Sites , Bungarotoxins/chemistry , Calorimetry , Circular Dichroism , Cloning, Molecular , Crystallography, X-Ray , Drug Design , In Situ Hybridization , Ions , Ligands , Models, Molecular , Molecular Sequence Data , Mollusca , Phylogeny , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal Transduction , Temperature
9.
Neuron ; 41(6): 907-14, 2004 Mar 25.
Article in English | MEDLINE | ID: mdl-15046723

ABSTRACT

Nicotinic acetylcholine receptors are prototypes for the pharmaceutically important family of pentameric ligand-gated ion channels. Here we present atomic resolution structures of nicotine and carbamylcholine binding to AChBP, a water-soluble homolog of the ligand binding domain of nicotinic receptors and their family members, GABAA, GABAC, 5HT3 serotonin, and glycine receptors. Ligand binding is driven by enthalpy and is accompanied by conformational changes in the ligand binding site. Residues in the binding site contract around the ligand, with the largest movement in the C loop. As expected, the binding is characterized by substantial aromatic and hydrophobic contributions, but additionally there are close contacts between protein oxygens and positively charged groups in the ligands. The higher affinity of nicotine is due to a main chain hydrogen bond with the B loop and a closer packing of the aromatic groups. These structures will be useful tools for the development of new drugs involving nicotinic acetylcholine receptor-associated diseases.


Subject(s)
Acetylcholine/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Ion Channel Gating/physiology , Nicotinic Agonists/metabolism , Animals , Binding Sites/drug effects , Binding Sites/physiology , Carbachol/metabolism , Carrier Proteins/drug effects , Crystallography, X-Ray , Ion Channel Gating/drug effects , Ligands , Lymnaea , Models, Molecular , Molecular Conformation , Nervous System/metabolism , Nicotine/metabolism , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/physiology , Synaptic Transmission/drug effects , Synaptic Transmission/physiology
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