RESUMO
The Xrcc3 protein, which is required for the homologous recombinational repair of damaged DNA, forms a complex with the Rad51C protein in human cells. Mutations in either the Xrcc3 or Rad51C gene cause extreme sensitivity to DNA-damaging agents and generate the genomic instability frequently found in tumors. In the present study, we found that the Xrcc3 segment containing amino acid residues 63-346, Xrcc3(63-346), is the Rad51C-binding region. Biochemical analyses revealed that Xrcc3(63-346) forms a complex with Rad51C, and the Xrcc3(63-346)- Rad51C complex possesses ssDNA and dsDNA binding abilities comparable to those of the full-length Xrcc3-Rad51C complex. Based on the structure of RecA, which is thought to be the ancestor of Xrcc3, six Xrcc3 point mutants were designed. Two-hybrid and biochemical analyses of the Xrcc3 point mutants revealed that Tyr139 and Phe249 are essential amino acid residues for Rad51C binding. Superposition of the Xrcc3 Tyr139 and Phe249 residues on the RecA structure suggested that Tyr139 may function to ensure proper folding and Phe249 may be important to constitute the Rad51C-binding interface in Xrcc3.
Assuntos
Aminoácidos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Aminoácidos/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Humanos , Dados de Sequência Molecular , Mutação , Fenilalanina/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Tirosina/genéticaRESUMO
The Rad51B, Rad51C, Rad51D and Xrcc2 proteins are Rad51 paralogs, and form a complex (BCDX2 complex) in mammalian cells. Mutant cells defective in any one of the Rad51-paralog genes exhibit spontaneous genomic instability and extreme sensitivity to DNA-damaging agents, due to inefficient recombinational repair. Therefore, the Rad51 paralogs play important roles in the maintenance of genomic integrity through recombinational repair. In the present study, we examined the DNA-binding preference of the human BCDX2 complex. Competitive DNA-binding assays using seven types of DNA substrates, single-stranded DNA (ssDNA), double-stranded DNA, 5'- and 3'-tailed duplexes, nicked duplex DNA, Y-shaped DNA and a synthetic Holliday junction, revealed that the BCDX2 complex preferentially bound to the two DNA substrates with branched structures (the Y-shaped DNA and the synthetic Holliday junction). Furthermore, the BCDX2 complex catalyzed the strand-annealing reaction between a long linear ssDNA (1.2 kb in length) and its complementary circular ssDNA. These properties of the BCDX2 complex may be important for its roles in the maintenance of chromosomal integrity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , DNA/química , DNA Cruciforme/metabolismo , DNA de Cadeia Simples/metabolismo , Substâncias Macromoleculares , Conformação de Ácido NucleicoRESUMO
In Saccharomyces cerevisiae, the Hop2 protein forms a complex with the Mnd1 protein and is required for the alignment of homologous chromosomes during meiosis, probably through extensive homology matching between them. The Rad51 and Dmc1 proteins, the eukaryotic RecA orthologs, promote strand exchange and may function in the extensive matching of homology within paired DNA molecules. In the present study, we purified the human TBPIP/Hop2-Mnd1 complex and found that it significantly stimulates the Dmc1- and Rad51-mediated strand exchange. The human Hop2-Mnd1 complex preferentially binds to a three-stranded DNA branch, which mimics the strand-exchange intermediate. These findings are consistent with genetic data, which showed that the Hop2 and Mnd1 proteins are required for homology matching between homologous chromosomes. Therefore, the human TBPIP/Hop2-Mnd1 complex may ensure proper pairing between homologous chromosomes through its stimulation of strand exchange during meiosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Recombinação Genética , Transativadores/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Complexos Multiproteicos , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Proteínas de Ligação a Fosfato , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genéticaRESUMO
In meiosis, homologous recombination preferentially occurs between homologous chromosomes rather than between sister chromatids, which is opposite to the bias of mitotic recombinational repair. The TBPIP/HOP2 protein is a factor that ensures the proper pairing of homologous chromosomes during meiosis. In the present study, we found that the purified mouse TBPIP/HOP2 protein stimulated homologous pairing catalyzed by the meiotic DMC1 recombinase in vitro. In contrast, TBPIP/HOP2 did not stimulate homologous pairing by RAD51, which is another homologous pairing protein acting in both meiotic and mitotic recombination. The positive effect of TBPIP/HOP2 in the DMC1-mediated homologous pairing was only observed when TBPIP/HOP2 first binds to double-stranded DNA, not to single-stranded DNA, before the initiation of the homologous pairing reaction. Deletion analyses revealed that the C-terminal basic region of TBPIP/HOP2 is required for efficient DNA binding and is also essential for its homologous pairing stimulation activity. Therefore, these results suggest that TBPIP/HOP2 directly binds to DNA and functions as an activator for DMC1 during the homologous pairing step in meiosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Adenosina Trifosfatases/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas de Ligação a Fosfato , Ligação Proteica , Rad51 Recombinase , Recombinação Genética , Transativadores/genéticaRESUMO
The human Dmc1 protein, a RecA/Rad51 homolog, is a meiosis-specific DNA recombinase that catalyzes homologous pairing. RecA and Rad51 form helical filaments, while Dmc1 forms an octameric ring. In the present study, we crystallized the full-length human Dmc1 protein and solved the structure of the Dmc1 octameric ring. The monomeric structure of the Dmc1 protein closely resembled those of the human and archaeal Rad51 proteins. In addition to the polymerization motif that was previously identified in the Rad51 proteins, we found another hydrogen bonding interaction at the polymer interface, which could explain why Dmc1 forms stable octameric rings instead of helical filaments. Mutagenesis studies identified the inner and outer basic patches that are important for homologous pairing. The inner patch binds both single-stranded and double-stranded DNAs, while the outer one binds single-stranded DNA. Based on these results, we propose a model for the interaction of the Dmc1 rings with DNA.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , DNA/química , Meiose/genética , Homologia de Sequência , Animais , Sítios de Ligação/genética , Cristalografia por Raios X , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Peso Molecular , Conformação Proteica , Estrutura Terciária de Proteína/genética , Recombinação Genética/genéticaRESUMO
The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.