RESUMEN
DNA strand exchange by serine recombinases has been proposed to occur by a large-scale rotation of halves of the recombinase tetramer. Here we provide the first direct physical evidence for the subunit rotation mechanism for the Hin serine invertase. Single-DNA looping assays using an activated mutant (Hin-H107Y) reveal specific synapses between two hix sites. Two-DNA "braiding" experiments, where separate DNA molecules carrying a single hix are interwound, show that Hin-H107Y cleaves both hix sites and mediates multi-step rotational relaxation of the interwinding. The variable numbers of rotations in the DNA braid experiments are in accord with data from bulk experiments that follow DNA topological changes accompanying recombination by the hyperactive enzyme. The relatively slow Hin rotation rates, combined with pauses, indicate considerable rotary friction between synapsed subunit pairs. A rotational pausing mechanism intrinsic to serine recombinases is likely to be crucial for DNA ligation and for preventing deleterious DNA rearrangements.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Nucleotidiltransferasas/metabolismo , ADN Superhelicoidal/metabolismo , Recombinación Genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , ADN Nucleotidiltransferasas/química , ADN Nucleotidiltransferasas/genética , ADN Superhelicoidal/química , Proteínas de Unión al ADN/metabolismo , Factor Proteico para Inverción de Estimulación/metabolismo , Modelos Químicos , Modelos Genéticos , Modelos Moleculares , Conformación de Ácido Nucleico , Plásmidos/genética , Mutación Puntual , Conformación Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotación , Salmonella/enzimologíaRESUMEN
Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism. DOI:http://dx.doi.org/10.7554/eLife.01211.001.
Asunto(s)
Proteínas Bacterianas/química , ADN Nucleotidiltransferasas/química , Elementos de Facilitación Genéticos , Factor Proteico para Inverción de Estimulación/química , Regulación Bacteriana de la Expresión Génica , Salmonella enterica/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , ADN Superhelicoidal , Escherichia coli/genética , Escherichia coli/metabolismo , Factor Proteico para Inverción de Estimulación/genética , Factor Proteico para Inverción de Estimulación/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación Genética , Salmonella enterica/metabolismoRESUMEN
Two new reports on serine recombinases, one of a crystal snapshot in an alternate rotational conformer poised for DNA cleavage (Keenholtz et al., 2011), and a second employing single-DNA molecule approaches (Bai et al., 2011), provide strong support for the subunit rotation model for exchanging DNA strands.
RESUMEN
Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the 'D-helix' region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange.