RESUMEN
Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Recombinación Genética/genética , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/deficiencia , Proteínas de Homeodominio/química , Ratones , Modelos Genéticos , Mutación/genéticaAsunto(s)
Bacterias/genética , Bacterias/metabolismo , Reparación del ADN , ADN Bacteriano/metabolismo , Antígenos Nucleares/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Daño del ADN , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Autoantígeno KuRESUMEN
Recent studies in Escherichia coli indicate that the interconversion of DNA replication fork and Holliday junction structures underpins chromosome duplication and helps secure faithful transmission of the genome from one generation to the next. It facilitates interplay between DNA replication, recombination and repair, and provides means to rescue replication forks stalled by lesions in or on the template DNA. Insight into how this interconversion may be catalysed has emerged from genetic, biochemical and structural studies of RecG protein, a member of superfamily 2 of DNA and RNA helicases. We describe how a single molecule of RecG might target a branched DNA structure and translocate a single duplex arm to drive branch migration of a Holliday junction, interconvert replication fork and Holliday junction structures and displace the invading strand from a D loop formed during recombination at a DNA end. We present genetic evidence suggesting how the latter activity may provide an efficient pathway for the repair of DNA double-strand breaks that avoids crossing over, thus facilitating chromosome segregation at cell division.
Asunto(s)
Segregación Cromosómica/fisiología , ADN Helicasas/química , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Proteínas de Escherichia coli/química , Recombinación Genética/fisiología , ADN/metabolismo , ADN Helicasas/metabolismo , ADN Cruciforme/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismoRESUMEN
Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA-binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved.
Asunto(s)
Antígenos Nucleares/química , Bacteriófago mu/química , ADN Helicasas , Proteínas de Unión al ADN/química , Proteínas Virales/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Bacteriófago mu/genética , Sitios de Unión , ADN/metabolismo , Dimerización , Células Eucariotas/metabolismo , Evolución Molecular , Haemophilus influenzae/química , Haemophilus influenzae/genética , Humanos , Autoantígeno Ku , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Retroelementos/genética , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis. These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.