ABSTRACT
The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice.
Subject(s)
Oryza/enzymology , Plant Proteins/metabolism , Recombinases/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , DNA/metabolism , Molecular Sequence Data , Plant Proteins/isolation & purification , Plant Proteins/ultrastructure , Recombinases/isolation & purification , Recombinases/ultrastructure , Sequence AlignmentABSTRACT
The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1-single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Recombinases/metabolism , Recombination, Genetic , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/ultrastructure , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/ultrastructure , DNA Helicases/isolation & purification , DNA Helicases/ultrastructure , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/ultrastructure , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/ultrastructure , Humans , Nuclear Proteins/isolation & purification , Nuclear Proteins/ultrastructure , Rad51 Recombinase/metabolism , Recombinases/chemistry , Recombinases/ultrastructureABSTRACT
UNLABELLED: TopoICE-R is a three-dimensional visualization and manipulation software for solving 2-string tangle equations and can be used to model the topology of DNA bound by proteins such as recombinases and topoisomerases. AVAILABILITY: This software, manual and example files are available at www.knotplot.com/download for Linux, Windows and Mac.
Subject(s)
DNA/chemistry , Models, Chemical , Models, Molecular , Recombinases/chemistry , Recombination, Genetic , Software , User-Computer Interface , Algorithms , Binding Sites , Computer Graphics , Computer Simulation , DNA/ultrastructure , Imaging, Three-Dimensional/methods , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Recombinases/ultrastructureABSTRACT
The rate of annealing of long linear complementary single-stranded (ss) DNAs can be increased greatly by certain DNA-binding proteins including the herpes simplex virus type 1 ICP8 SSB/recombinase. Using electron microscopy, we have investigated the DNA-protein structures involved in ICP8-mediated DNA annealing. We show that the formation of superhelical ICP8-ssDNA filaments is required for annealing. Two superhelices interact with each other to form a coiled-coil, which is the intermediate in annealing. In this process, the superhelices likely rotate and translocate relative to each other. Psoralen/UV photocrosslinking studies revealed that meta-stable contacts form at sites of limited sequence homology during the annealing. Partial proteolysis of ICP8 in the protein-ssDNA complexes showed that Mg2+ induces conformational changes in the N-terminal region (amino acid residues 1-305) of ICP8. In addition to Mg2+, Ca2+ and, to a significantly lesser extent, Cu2+ and Mn2+, were found to induce superhelix formation of the ICP8-ssDNA filament and to facilitate annealing. Mechanisms for how the coiled-coil structures facilitate annealing are discussed.
Subject(s)
DNA, Single-Stranded , Nucleic Acid Conformation , Protein Conformation , Recombinases/metabolism , Viral Proteins/metabolism , Animals , Cross-Linking Reagents/metabolism , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/ultrastructure , DNA-Binding Proteins , Ficusin/metabolism , Magnesium/metabolism , Models, Molecular , Protein Renaturation , Recombinases/ultrastructure , Viral Proteins/ultrastructureABSTRACT
The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed.
