RESUMEN
Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Telomerasa/deficiencia , Replicación del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endonucleasas de ADN Solapado/genética , Viabilidad Microbiana , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telómero/metabolismo , Homeostasis del TelómeroRESUMEN
The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.
Asunto(s)
Carcinógenos/toxicidad , Transformación Celular Neoplásica , Resistencia a Medicamentos/genética , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Mutagénesis , Secuencia de Bases/fisiología , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Células Cultivadas , Daño del ADN , Resistencia a Medicamentos/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Humanos , Melanoma/etiología , Melanoma/genética , Mutagénesis/efectos de los fármacos , Mutagénesis/genética , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/genética , Rayos Ultravioleta , Melanoma Cutáneo MalignoRESUMEN
Chromatin remodeling complexes are essential for gene expression programs that coordinate cell function with metabolic status. However, how these remodelers are integrated in metabolic stability pathways is not well known. Here, we report an expansive genetic screen with chromatin remodelers and metabolic regulators in Saccharomyces cerevisiae. We found that, unlike the SWR1 remodeler, the INO80 chromatin remodeling complex is composed of multiple distinct functional subunit modules. We identified a strikingly divergent genetic signature for the Ies6 subunit module that links the INO80 complex to metabolic homeostasis. In particular, mitochondrial maintenance is disrupted in ies6 mutants. INO80 is also needed to communicate TORC1-mediated signaling to chromatin, as ino80 mutants exhibit defective transcriptional profiles and altered histone acetylation of TORC1-responsive genes. Furthermore, comparative analysis reveals subunits of INO80 and mTORC1 have high co-occurrence of alterations in human cancers. Collectively, these results demonstrate that the INO80 complex is a central component of metabolic homeostasis that influences histone acetylation and may contribute to disease when disrupted.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Acetilación , Regulación Fúngica de la Expresión Génica , Inestabilidad Genómica/genética , Homeostasis/genética , Redes y Vías Metabólicas/genética , Organismos Modificados Genéticamente , Procesamiento Proteico-Postraduccional/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81-Mms4/EME1, Slx1-Slx4/BTBD12/MUS312, XPF-ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.
Asunto(s)
Endonucleasas/metabolismo , Eucariontes/genética , Recombinación Genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Cruciforme/genética , Endonucleasas/genética , Eucariontes/enzimología , HumanosRESUMEN
Carcinogenic insult, such as UV light exposure, creates DNA lesions that evolve into mutations if left unrepaired. These resulting mutations can contribute to carcinogenesis and drive malignant phenotypes. Susceptibility to carcinogens (i.e., the propensity to form a carcinogen-induced DNA lesion) is regulated by both genetic and epigenetic factors. Importantly, carcinogen susceptibility is a critical contributor to cancer mutagenesis. It is known that mutations can be prevented by tumor suppressor regulation of DNA damage response pathways; however, their roles carcinogen susceptibility have not yet been reported. In this study, we reveal that the retinoblastoma (RB1) tumor suppressor regulates UV susceptibility across broad regions of the genome. In particular, centromere and telomere-proximal regions exhibit significant increases in UV lesion susceptibility when RB1 is deleted. Several cancer-related genes are located within genomic regions of increased susceptibility, including telomerase reverse transcriptase, TERT, thereby accelerating mutagenic potential in cancers with RB1 pathway alterations. These findings reveal novel genome stability mechanisms of a tumor suppressor and uncover new pathways to accumulate mutations during cancer evolution.
Asunto(s)
Carcinogénesis , Carcinógenos/farmacología , Neoplasias , Proteínas de Unión a Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genética , Sistemas CRISPR-Cas , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Línea Celular , Técnicas de Inactivación de Genes , Predisposición Genética a la Enfermedad/genética , Humanos , Mutación/genética , Neoplasias/genética , Neoplasias/patología , Oncogenes/genéticaRESUMEN
Exposure to the ultraviolet (UV) radiation in sunlight creates DNA lesions, which if left unrepaired can induce mutations and contribute to skin cancer. The two most common UV-induced DNA lesions are the cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), both of which can initiate mutations. Interestingly, mutation frequency across the genomes of many cancers is heterogenous with significant increases in heterochromatin. Corresponding increases in UV lesion susceptibility and decreases in repair are observed in heterochromatin versus euchromatin. However, the individual contributions of CPDs and 6-4PPs to mutagenesis have not been systematically examined in specific genomic and epigenomic contexts. In this study, we compared genome-wide maps of 6-4PP and CPD lesion abundances in primary cells and conducted comprehensive analyses to determine the genetic and epigenetic features associated with susceptibility. Overall, we found a high degree of similarity between 6-4PP and CPD formation, with an enrichment of both in heterochromatin regions. However, when examining the relative levels of the two UV lesions, we found that bivalent and Polycomb-repressed chromatin states were uniquely more susceptible to 6-4PPs. Interestingly, when comparing UV susceptibility and repair with melanoma mutation frequency in these regions, disparate patterns were observed in that susceptibility was not always inversely associated with repair and mutation frequency. Functional enrichment analysis hint at mechanisms of negative selection for these regions that are essential for cell viability, immune function and induce cell death when mutated. Ultimately, these results reveal both the similarities and differences between UV-induced lesions that contribute to melanoma.
Asunto(s)
Reparación del ADN , Epigénesis Genética/efectos de la radiación , Melanoma/genética , Mutación , Neoplasias Cutáneas/genética , Rayos Ultravioleta/efectos adversos , Daño del ADN , Bases de Datos Genéticas , Eucromatina/química , Eucromatina/metabolismo , Eucromatina/efectos de la radiación , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Genoma Humano/efectos de la radiación , Heterocromatina/química , Heterocromatina/metabolismo , Heterocromatina/efectos de la radiación , Histonas/genética , Histonas/metabolismo , Humanos , Melanoma/etiología , Melanoma/metabolismo , Melanoma/patología , Mutagénesis , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Cultivo Primario de Células , Dímeros de Pirimidina/agonistas , Dímeros de Pirimidina/metabolismo , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3' flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3' flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Reparación del ADN por Recombinación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación GenéticaRESUMEN
Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.