ABSTRACT
Homologous recombination (HR) is a template-driven repair pathway that mends DNA double-stranded breaks (DSBs), and thus helps to maintain genome stability. The RAD51 recombinase facilitates DNA joint formation during HR, but to accomplish this task, RAD51 must be loaded onto the single-stranded DNA. DSS1, a candidate gene for split hand/split foot syndrome, provides the ability to recognize RPA-coated ssDNA to the tumor suppressor BRCA2, which is complexed with RAD51. Together BRCA2-DSS1 displace RPA and load RAD51 onto the ssDNA. In addition, the BRCA2 interacting protein BCCIP normally colocalizes with chromatin bound BRCA2, and upon DSB induction, RAD51 colocalizes with BRCA2-BCCIP foci. Down-regulation of BCCIP reduces DSB repair and disrupts BRCA2 and RAD51 foci formation. While BCCIP is known to interact with BRCA2, the relationship between BCCIP and RAD51 is not known. In this study, we investigated the biochemical role of the ß-isoform of BCCIP in relation to the RAD51 recombinase. We demonstrate that BCCIPß binds DNA and physically and functionally interacts with RAD51 to stimulate its homologous DNA pairing activity. Notably, this stimulatory effect is not the result of RAD51 nucleoprotein filament stabilization; rather, we demonstrate that BCCIPß induces a conformational change within the RAD51 filament that promotes release of ADP to help maintain an active presynaptic filament. Our findings reveal a functional role for BCCIPß as a RAD51 accessory factor in HR.
Subject(s)
Adenosine Diphosphate/metabolism , Base Pairing , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Homologous Recombination , Nuclear Proteins/metabolism , Rad51 Recombinase/metabolism , Adenosine Triphosphate/metabolism , Calcium-Binding Proteins/chemistry , Cell Cycle Proteins/chemistry , DNA Repair , Humans , Hydrolysis , Nuclear Proteins/chemistry , Protein Binding , Protein Conformation , Protein Isoforms , Protein MultimerizationABSTRACT
The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.
Subject(s)
Entamoeba histolytica/enzymology , Homologous Recombination , Protozoan Proteins/metabolism , Rad51 Recombinase/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Adenosine Triphosphate/metabolism , Calcium/metabolism , Carrier Proteins , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Repair , Enzyme Activation , Hydrolysis , Nucleic Acid Conformation , Plasmids/genetics , Protein Binding/drug effects , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Rad51 Recombinase/genetics , Rad51 Recombinase/isolation & purification , Recombinant Proteins , Substrate SpecificityABSTRACT
Survivin belongs to the family of inhibitor of apoptosis proteins (IAP) and is present in most cancers while being below detection limits in most terminally differentiated adult tissues, making it an attractive protein to target for diagnostic and, potentially, therapeutic roles. Sub-100 nm poly(propargyl acrylate) (PA) particles were surface modified through the copper-catalyzed azide/alkyne cycloaddition of an azide-terminated survivin ligand derivative (azTM) originally proposed by Abbott Laboratories and speculated to bind directly to survivin (protein) at its dimer interface. Using affinity pull-down studies, it was determined that the PA/azTM nanoparticles selectively bind survivin and the particles can enhance apoptotic cell death in glioblastoma cell lines and other survivin over-expressing cell lines such as A549 and MCF7 relative to cells incubated with the original Abbott-derived small molecule inhibitor.
Subject(s)
Acrylates/chemistry , Apoptosis , Azides/chemistry , Inhibitor of Apoptosis Proteins/chemistry , Nanoparticles/chemistry , Neoplasm Proteins/chemistry , Polymers/chemistry , Apoptosis/physiology , Azides/pharmacology , Catalysis , Cell Line, Tumor , Copper/chemistry , Cycloaddition Reaction , Humans , Inhibitor of Apoptosis Proteins/metabolism , Inhibitor of Apoptosis Proteins/pharmacology , Ligands , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolismABSTRACT
Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.
Subject(s)
Calcium/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Entamoeba histolytica/metabolism , Homologous Recombination , Protozoan Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , DNA-Binding Proteins/genetics , Mice , Protozoan Proteins/geneticsABSTRACT
During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1(K132R)) or alanine (hDMC1(K132A)). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1(K132R) and hDMC1(K132A) variants were devoid of ATP hydrolysis. The hDMC1(K132R) variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1(K132R) variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1(K132A) variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.
Subject(s)
Cell Cycle Proteins/chemistry , DNA-Binding Proteins/chemistry , Lysine/chemistry , Adenosine Triphosphate/metabolism , Alanine/genetics , Amino Acid Motifs , Amino Acid Sequence , Arginine/genetics , Binding Sites , Calcium/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Hydrolysis , Lysine/genetics , Molecular Sequence Data , Mutation, Missense , Protein Structure, TertiaryABSTRACT
Meiotic homologous recombination in Saccharomyces cerevisiae involves formation of nucleoprotein filaments of Rad51 and Dmc1 that mediate DNA strand exchange between homologous chromosomes. The Mei5-Sae3 protein complex functions as a recombination mediator to promote nucleation of the Dmc1 recombinase onto replication protein A-coated single-stranded DNA. Here, we have expressed and purified the Mei5 protein, Sae3 protein and the Mei5-Sae3 complex for biochemical studies. We show the Mei5-Sae3 complex preferentially binds a fork-like DNA substrate to 3' overhanging DNA, single-stranded DNA or double-stranded DNA. We demonstrate that Mei5 confers DNA binding activity to the Mei5-Sae3 complex. We determined Mei5-Sae3 interacts with the Rad51 recombinase through the N-terminal domain of Mei5. Unlike Rad52, Mei5-Sae3 lacks recombination mediator activity for Rad51. Importantly, we find that the Mei5-Sae3 complex does not harbor single-strand DNA annealing activity. These properties of the Mei5-Sae3 complex distinguishes it from the Rad52 protein, which serves as the mediator of Rad51 and is involved in the single-strand DNA annealing pathway of homologous recombination.
