RESUMEN
BRCT-containing protein 1 (Brc1) is a multi-BRCT (BRCA1 carboxyl terminus) domain protein in Schizosaccharomyces pombe that is required for resistance to chronic replicative stress, but whether this reflects a repair or replication defect is unknown and the subject of this study. We show that brc1Δ cells are significantly delayed in recovery from replication pausing, though this does not activate a DNA damage checkpoint. DNA repair and recombination protein Rad52 is a homologous recombination protein that loads the Rad51 recombinase at resected double-stranded DNA (dsDNA) breaks and is also recruited to stalled replication forks, where it may stabilize structures through its strand annealing activity. Rad52 is required for the viability of brc1Δ cells, and brc1Δ cells accumulate Rad52 foci late in S phase that are potentiated by replication stress. However, these foci contain the single-stranded DNA (ssDNA) binding protein RPA, but not Rad51 or γH2A. Further, these foci are not associated with increased recombination between repeated sequences, or increased post-replication repair. Thus, these Rad52 foci do not represent sites of recombination. Following the initiation of DNA replication, the induction of these foci by replication stress is suppressed by defects in origin recognition complex (ORC) function, which is accompanied by loss of viability and severe mitotic defects. This suggests that cells lacking Brc1 undergo an ORC-dependent rescue of replication stress, presumably through the firing of dormant origins, and this generates RPA-coated ssDNA and recruits Rad52. However, as Rad51 is not recruited, and the checkpoint effector kinase Chk1 is not activated, these structures must not contain the unprotected primer ends found at sites of DNA damage that are required for recombination and checkpoint activation.
Asunto(s)
Replicación del ADN , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Estrés Fisiológico , Replicación del ADN/efectos de los fármacos , ADN Ribosómico/metabolismo , Hidroxiurea/farmacología , Mutagénesis/efectos de los fármacos , Mutagénesis/genética , Mutación/genética , Complejo de Reconocimiento del Origen/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Recombinación Genética/genética , Proteína de Replicación A/metabolismo , Fase S/efectos de los fármacos , Schizosaccharomyces/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
Smc5/6 is a structural maintenance of chromosomes complex, related to the cohesin and condensin complexes. Recent studies implicate Smc5/6 as being essential for homologous recombination. Each gene is essential, but hypomorphic alleles are defective in the repair of a diverse array of lesions. A particular allele of smc6 (smc6-74) is suppressed by overexpression of Brc1, a six-BRCT domain protein that is required for DNA repair during S-phase. This suppression requires the postreplication repair (PRR) protein Rhp18 and the structure-specific endonucleases Slx1/4 and Mus81/Eme1. However, we show here that the contribution of Rhp18 is via a novel pathway that is independent of PCNA ubiquitination and PRR. Moreover, we identify Exo1 as an additional nuclease required for Brc1-mediated suppression of smc6-74, independent of mismatch repair. Further, the Apn2 endonuclease is required for the viability of smc6 mutants without extrinsic DNA damage, although this is not due to a defect in base excision repair. Several nucleotide excision repair genes are similarly shown to ensure viability of smc6 mutants. The requirement for excision factors for the viability of smc6 mutants is consistent with an inability to respond to spontaneous lesions by Smc5/6-dependent recombination.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Secuencia de Bases , Reparación del ADN/genética , ADN de Hongos/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Genes Fúngicos , Mutación , Recombinación Genética , Schizosaccharomyces/crecimiento & desarrolloRESUMEN
The structural maintenance of chromosome (SMC) proteins are key elements in controlling chromosome dynamics. In eukaryotic cells, three essential SMC complexes have been defined: cohesin, condensin, and the Smc5/6 complex. The latter is essential for DNA damage responses; in its absence both repair and checkpoint responses fail. In fission yeast, the UV-C and ionizing radiation (IR) sensitivity of a specific hypomorphic allele encoding the Smc6 subunit, rad18-74 (renamed smc6-74), is suppressed by mild overexpression of a six-BRCT-domain protein, Brc1. Deletion of brc1 does not result in a hypersensitivity to UV-C or IR, and thus the function of Brc1 relative to the Smc5/6 complex has remained unclear. Here we show that brc1Delta cells are hypersensitive to a range of radiomimetic drugs that share the feature of creating lesions that are an impediment to the completion of DNA replication. Through a genetic analysis of brc1Delta epistasis and by defining genes required for Brc1 to suppress smc6-74, we find that Brc1 functions to promote recombination through a novel postreplication repair pathway and the structure-specific nucleases Slx1 and Mus81. Activation of this pathway through overproduction of Brc1 bypasses a repair defect in smc6-74, reestablishing resolution of lesions by recombination.
