BOCCO JOSE LUIS
Congresos y reuniones científicas
Título:
Identification and Validation of a Novel Translesion DNA Synthesis Inhibitor Through a Western-Blot Based Screening Platform
Autor/es:
VILLAFAÑEZ F.; GARCÍA A.; MANSILLA S.; PANSA M.F.; CARBAJOSA S.; BOCCO J.L.; GOTTIFREDI V.; SORIA G.
Lugar:
Buenos Aires
Reunión:
Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017
Institución organizadora:
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Resumen:
Translesion DNA synthesis (TLS) is a DNA damage tolerance process that employs specialized polymerases to bypass DNA damage during replication. Several lines of evidence suggest that TLS inhibition would be crucial in the homologous recombination (HR) deficient context of certain types of cancers (i.e. BRCA-/- breast and ovarian tumors). However, the limitation to explore such type of therapeutic strategy is the lack of chemical inhibitors to target TLS. The main goal of this project is to identify specific inhibitors of TLS that can be used as ?a proof of concept? to induce selective toxicity in BRCA-deficient cells. Our rational is that since TLS polymerases recruitment to sites of DNA damage is a key step for TLS success, we can indirectly monitor TLS efficiency by studying two key markers:1) The mono-ubiquitylation of PCNA and 2) the accumulation of a TLS polymerase into replication foci. Herein, we developed a screening platform to identify inhibitors of PCNA mono-ubiquitylation through a Western-Blot based method. From our first screening with an open source library of kinase inhibitors from GlaxoSmithKline we identified a number of hits that inhibit mono-ubiquitylation PCNA. Here we describe the validation of these hits using commercial inhibitors, cell cycle analysis, characterization of DNA damage markers and DNA combing assays to select targets that impair replication processivity after UV irradiation.Moreover, with the validated hits we performed proof of concept experiments in HR-deficient cells, unveiling a link between these pathways in the promotion of cell survival after UV.