RESUMEN
During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.
Asunto(s)
ADN/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión/genética , ADN/genética , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , ADN de Cadena Simple/genética , Recombinación Homóloga/genética , Recombinación Homóloga/fisiología , Humanos , Mutación/genética , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Estructura Secundaria de Proteína , Recombinasa Rad51/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMEN
Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.
Asunto(s)
Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , ADN/metabolismo , Complejos Multiproteicos/metabolismo , Multimerización de Proteína/fisiología , Factor de Transcripción YY1/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Portadoras/genética , Línea Celular , ADN/genética , ADN Helicasas/genética , Recombinación Homóloga/fisiología , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Unión Proteica/fisiología , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Transcripción Genética/fisiología , Factor de Transcripción YY1/genéticaRESUMEN
Local hyperthermia is an effective treatment modality to augment radio- and chemotherapy-based anti-cancer treatments. Although the effect of hyperthermia is pleotropic, recent experiments revealed that homologous recombination, a pathway of DNA repair, is directly inhibited by hyperthermia. The hyperthermia-induced DNA repair deficiency is enhanced by inhibitors of the cellular heat-shock response. Taken together, these results provide the rationale for the development of novel anti-cancer therapies that combine hyperthermia-induced homologous recombination deficiency with the systemic administration of drugs that specifically affect the viability of homologous recombination deficient cells and/or inhibit the heat-shock response, to locally sensitise cancer cells to DNA damaging agents.