Upon DNA damage cells activate a genetically controlled pathway, known as DNA damage checkpoint, which is devoted to helping cells cope with genomic lesions. Much of the molecular details underlying this response are now understood, but the integration between checkpoint factors and other cellular pathways are still mostly undefined. We are trying to identify new mechanisms involved in the maintenance of genome stability, using multiple parallel approaches in the genetically tractable model system Saccharomyces cerevisiae. 1. Identification of the substrates of checkpoint kinases. Taking advantage of a biotin ligase module, derived from E.coli, we generated a yeast strain which expresses a Rad53 protein, which contains also a biotin ligase activity. This chimeric protein is fully active in the checkpoint response and in the preservation of stalled replication forks. Diploid cells obtained by crossing this strain with a collection of yeast cells expressing individual genes tagged with a biotinilation motif, will be screened for targets of the Rad53 checkpoint kinase. 2. Identification of new genes required for the maintenance of genome integrity. Several mutations in genes known to be involved in the preservation of genome stability cause a variable level of phosphorylation of Rad53 in physiological growth conditions. This is likely due to a higher background level of endogenous genomic instability. Overexpression of yeast checkpoint proteins in such strains leads to cell lethality, probably due to the imposition of cell cycle arrest caused by the simultaneous presence of the mutation and a hypersensitive checkpoint pathway. We will take advantage of this strategy to perform an automated systematic search of a yeast gene deletion collection for mutants which are synthetically lethal when the checkpoint is overstimulated. 3. Systematic identification of new factors interacting with checkpoint factors. We are generating a yeast strain collection expressing checkpoint proteins as a two-hybrid bait. We also produced a yeast prey library which is expressed in cells of the opposite mating type. By large scale mating experiments and taking advantage of a fluorescent automated screening methodology, we will systematically search for yeast proteins physically interacting with all the factors involved in the checkpoint signal transduction cascade.
|Titolo:||Systematic approaches for the definition of the DNA damage response pathways in budding yeast|
MUZI FALCONI, MARCO (Corresponding)
|Data di pubblicazione:||31-mar-2009|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||Systematic approaches for the definition of the DNA damage response pathways in budding yeast / F. Lazzaro, A. Capra, D. Novarina, M. Giannattasio, P. Plevani, M. Muzi Falconi. ((Intervento presentato al 3. convegno Workshop on Systems level understanding of DNA damage responses tenutosi a Egmond aam Zee nel 2009.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|