RESUMO
Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.
Assuntos
Reparo do DNA/genética , Replicação do DNA/genética , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Archaeoglobus , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , DNA/genética , DNA/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Conformação Molecular , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Saccharomyces cerevisiae , Especificidade por Substrato/fisiologiaRESUMO
Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection. Expression of Nur77 in thymocytes and cell lines leads to apoptosis through a mechanism that remains unclear. In some cell lines, Nur77 was reported to act through a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release. However, we show here that Nur77-mediated apoptosis in thymocytes does not involve cytoplasmic cytochrome c release and cannot be rescued by Bcl-2. Microarray analysis shows that Nur77 induces many genes, including two novel genes (NDG1, NDG2) and known apoptotic genes FasL and TRAIL. Characterization of NDG1 and NDG2 indicates that NDG1 initiates a novel apoptotic pathway in a Bcl-2-independent manner. Thus Nur77-mediated apoptosis in T cells involves Bcl-2 independent transcriptional activation of several known and novel apoptotic pathways.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/fisiologia , Receptores de Esteroides/fisiologia , Linfócitos T/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Primers do DNA , Proteínas de Ligação a DNA/genética , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/fisiologia , Feminino , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Ratos , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Citoplasmáticos e Nucleares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Transcrição/genéticaRESUMO
To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.
