RESUMEN
Saccharomyces cerevisiae ribosomal DNA, the repeated region where rRNAs are synthesized by about 150 encoding units, hosts all the protein machineries responsible for the main DNA transactions such as replication, transcription and recombination. This and its repetitive nature make rDNA a unique and complex genetic locus compared to any other. All the different molecular machineries acting in this locus need to be accurately and finely controlled and coordinated and for this reason rDNA is one of the most impressive examples of highly complex molecular regulated loci. The region in which the large molecular complexes involved in rDNA activity and/or regulation are recruited is extremely small: that is, the 2.5 kb long intergenic spacer, interrupting each 35S RNA coding unit from the next. All S. cerevisiae RNA polymerases (I, II and III) transcribing the different genetic rDNA elements are recruited here; a sequence responsible for each rDNA unit replication, which needs its molecular apparatus, also localizes here; moreover, it is noteworthy that the rDNA replication proceeds almost unidirectionally because each replication fork is stopped in the so-called replication fork barrier. These localized fork blocking events induce, with a given frequency, the homologous recombination process by which cells maintain a high identity among the rDNA repeated units. Here, we describe the different processes involving the rDNA locus, how they influence each other and how these mutual interferences are highly regulated and coordinated. We propose that an rDNA conformation as a super-hub could help in optimizing the micro-environment for all basic DNA transactions.
Asunto(s)
Replicación del ADN/genética , ADN Ribosómico/genética , Recombinación Genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
S. cerevisiae ribosomal DNA (rDNA) locus hosts a series of highly complex regulatory machineries for RNA polymerase I, II and III transcription, DNA replication and units recombination, all acting in the Non Transcribed Spacers (NTSs) interposed between the repeated units by which it is composed. DNA topoisomerase I (Top1p) contributes, recruiting Sir2p, to the maintenance of transcriptional silencing occurring at the RNA Polymerase II cryptic promoters, located in the NTS region. In this paper we found that Fob1p presence is crucial for Top1p recruitment at NTS, allowing transcriptional silencing to be established and maintained. We also showed the role of Nsr1p in Top1p recruitment to rDNA locus. Our work allows to hypothesize that Nsr1p targets Top1p into the nucleolus while Fob1p is responsible for its preferential distribution at RFB.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Silenciador del Gen , Sitios Genéticos/genética , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética/genética , Replicación del ADN/genética , ARN Ribosómico/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
Ataxia telangiectasia is a rare autosomal recessive genome instability syndrome caused by mutations in the Ataxia Telangiectasia Mutated gene and characterized by a very high sensitivity to agents inducing double strand breaks such as ionizing radiation. In cells derived from ataxia telangiectasia patients a prominent enhancement of chromosomal aberrations is revealed as a consequence of this radiosensitivity characteristic, arising from defective DNA repair for a small fraction of breaks localized in the less accessible heterochromatin. Moreover, the signaling mediated by ataxia telangiectasia protein kinase also modifies chromatin structure. Even if there is a lot of knowledge concerning biochemical aspects of repair of double strand breaks, no conclusive results on radiosensitivity of structurally- and functionally-different chromatin are available, particularly in ataxia telangiectasia cells. Thus, a wild-type cell line and two ataxia telangiectasia patient derived ones could represent a suitable model to study the possible relationship between chromatin conformation and sensitivity to ionizing radiation. In this context, the effects of both cytosine arabinoside, an inhibitor of DNA repair synthesis, and trichostatin A, a histone deacetylase inhibitor, were tested in normal and ataxia telangiectasia lymphoblastoid cell lines carrying different mutation in the Ataxia Telangiectasia Mutated gene. The response to both inhibitors was investigated analyzing two endpoints, namely, chromosomal aberrations and the removal of DNA lesions by Comet assay, after exposure to X-rays. Results obtained suggest that the modulation of chromatin structure by trichostatin A leading to a more open conformation, decreases radiation-induced chromosomal aberrations in ataxia telangiectasia cells. The reduction in chromosomal instability can be attributed to an enhancement in DNA repair occurring in the presence of the histone deacetylase inhibitor, as its abolishment by the known inhibitor of DNA repair synthesis cytosine arabinoside clearly demonstrates. Data obtained could indicate a pivotal role of chromatin conformation in the radiosensitivity of ataxia telangiectasia cells.