RESUMEN
ATM(Tel1) and ATR(Rad3) checkpoint kinases phosphorylate the C-terminus of histone H2AX (H2A in yeasts) in chromatin flanking DNA damage, establishing a recruitment platform for checkpoint and repair proteins. Phospho-H2A/X (gammaH2A/X)-binding proteins at double-strand breaks (DSBs) have been characterized, but those required for replication stress responses are unknown. Here, we present genetic, biochemical, small angle X-ray scattering (SAXS), and X-ray structural studies of the Schizosaccharomyces pombe Brc1, a 6-BRCT-domain protein that is structurally related to Saccharomyces cerevisiae Rtt107 and mammalian PTIP. Brc1 binds gammaH2A to form spontaneous and DNA damage-induced nuclear foci. Spontaneous Brc1 foci colocalize with ribosomal DNA repeats, a region prone to fork pausing and genomic instability, whereas DNA damage-induced Brc1 foci colocalize with DSB response factors. gammaH2A binding is critical for Brc1 function. The 1.45 A resolution crystal structure of Brc1-gammaH2A complex shows how variable BRCT insertion loops sculpt tandem-BRCT phosphoprotein-binding pockets to facilitate unique phosphoprotein-interaction specificities, and unveils an acidic DNA-mimicking Brc1 surface. From these results, Brc1 docking to gammaH2A emerges as a critical chromatin-specific response to replication-associated DNA damage.
Asunto(s)
Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cristalografía por Rayos X , Daño del ADN , Histonas/genética , Modelos Moleculares , Conformación Proteica , Fase S , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Mms1 and Mms22 are subunits of an Rtt101-based E3 ubiquitin ligase required for replication of damaged DNA templates in Saccharomyces cerevisiae. The function and evolutionary conservation of this DNA repair module are unknown. Here we report the characterization of an Mms1 ortholog in Schizosaccharomyces pombe. Fission yeast Mms1 was discovered through its physical association with S. pombe Mms22 (also known as Mus7). Loss of S. pombe Mms1 results in the accumulation of spontaneous DNA damage, mitotic delay, and hypersensitivity to genotoxins such as camptothecin that perturb replisome progression. Homologous recombination repair proteins Rhp51 and Rad22 (Rad51 and Rad52 orthologs, respectively) are critical for survival in the absence of Mms1; however, there is no such requirement for Mus81-Eme1 Holliday junction resolvase that is essential for recovery from broken replication forks. Mms1 and Mms22 mutants share similar phenotypes and are genetically epistatic under unperturbed growth conditions and following exposure to genotoxins. From these data we conclude that an evolutionary conserved Mms1-Mms22 complex is required for replication of damaged DNA in fission yeast.
Asunto(s)
Replicación del ADN , ADN de Hongos/genética , Genoma Fúngico , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Daño del ADN , Reparación del ADN , Datos de Secuencia Molecular , Fenotipo , Unión Proteica , Schizosaccharomyces/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The faithful replication of the genome, coupled with the accurate repair of DNA damage, is essential for the maintenance of chromosomal integrity. The MMS22 gene of Saccharomyces cerevisiae plays an important but poorly understood role in preservation of genome integrity. Here we describe a novel gene in Schizosaccharomyces pombe that we propose is a highly diverged ortholog of MMS22. Fission yeast Mms22 functions in the recovery from replication-associated DNA damage. Loss of Mms22 results in the accumulation of spontaneous DNA damage in the S- and G2-phases of the cell cycle and elevated genomic instability. There are severe synthetic interactions involving mms22 and most of the homologous recombination proteins but not the structure-specific endonuclease Mus81-Eme1, which is required for survival of broken replication forks. Mms22 forms spontaneous nuclear foci and colocalizes with Rad22 in cells treated with camptothecin, suggesting that it has a direct role in repair of broken replication forks. Moreover, genetic interactions with components of the DNA replication fork suggest that Mms2 functions in the coordination of DNA synthesis following damage. We propose that Mms22 functions directly at the replication fork to maintain genomic integrity in a pathway involving Mus81-Eme1.
Asunto(s)
Replicación del ADN , ADN de Hongos/genética , Inestabilidad Genómica/fisiología , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Daño del ADN , Reparación del ADN , Fase G2/efectos de los fármacos , Fase G2/fisiología , Datos de Secuencia Molecular , Mutación/genética , Recombinación Genética , Fase S/efectos de los fármacos , Fase S/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The marriage between transducers of cell stress stimuli and their nuclear targets is likely to be achieved in part by some spatial-temporal compartmentalization of the relevant effectors. A candidate compartment for these events is the promyelocytic leukemia nuclear domain (PML-ND), within which are found numerous effectors of damage recognition, repair, and cell death. We predicted that the identification of PML-ND cargo proteins would clarify those biochemical pathways that straddle the recognition of cellular damage and cell fate. We now use mass spectrometry of peptides eluted from PML coprecipitates to demonstrate that the gamma 1 (gamma1) isoform of PLC associates with nuclear PML. Though thought to act primarily in the cytoplasm, we use biochemical fractionation combined with immunocytochemistry to verify the nuclear expression of PLC-gamma1 and its interaction with PML. These are the first data to show an interaction between endogenous levels of a phosphoinositide metabolizing protein and the biophysically labile PML-ND by mass spectrometry and add weight to the view that PML-NDs may act as tumor suppressors by sequestering mitogenic effectors.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosfolipasa C gamma/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Cromatografía en Gel , Proteínas Co-Represoras , Humanos , Espectrometría de Masas , Chaperonas Moleculares , Proteínas de Neoplasias/química , Proteínas Nucleares/química , Péptidos/química , Péptidos/metabolismo , Fosfolipasa C gamma/química , Proteína de la Leucemia Promielocítica , Factores de Transcripción/química , Proteínas Supresoras de Tumor/químicaRESUMEN
The polyglutamine diseases are characterized by expansion of triplet CAG repeats that encode polyglutamine tracts in otherwise unrelated proteins. One plausible explanation for the neurodegeneration of these disorders proposes that inclusions of such proteins sequester other significant nuclear proteins in inactive form. The present study shows that PML protein is sequestered by inclusions of the pathogenic mutant form of the polyglutamine protein ataxin-1 and that this sequestration removes from the nucleus the free 0.2-1 microm diameter PML nuclear domains (PML-NDs), together with at least one of their many cargo proteins (Sp100). The present study demonstrates that this sequestration can be effected equally by another nuclear protein, RED, which lacks a polyglutamine tract, but expresses a polar zipper repeat. The sequestered PML-NDs no longer respond to stress signals (heat shock or ionizing radiation) to which they are normally sensitive. In both cases, there is independent evidence that the cells initiate other responses to their injury (nuclear translocation of heat shock protein or generation of gamma-H2AX-rich nuclear foci, respectively). The data thus provide strong evidence that multiple species of nuclear inclusion functionally sequester PML-NDs. This mechanism is likely to distort cellular responses to injury of many different types.