Abstract 2960: A combined bioinformatics and proteomics approach identifies DNA repair factors regulated by the APC/C

Abstract

Abstract Our genome is constantly challenged by endogenous and exogenous factors that induce a variety of DNA lesions. DNA double strand breaks are considered to most toxic type of DNA damage, and cells have evolved powerful mechanisms that can detect and repair such lesions. Two main mechanisms responsible for repair of DNA double strand breaks are Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). The choice between these two types of DNA double strand break repair is strongly influenced by the cell cycle. Only cells in late S and G2 phase of the cell cycle can employ HR and this differential activity is underscored by a strict requirement for Cdk activity for HR. Mechanisms that enable HR during S-phase have been previously identified, but how exactly HR DNA repair is inactivated during the mitosis-to-G1 transition is poorly understood. Based on the observed strict cell-cycle regulation of HR repair, a role for the mitotic E3 ligase APC/C was suggested. Here we present a combined proteomics and bioinformatics approach to identify APC/C targets and reveal components of DNA double strand break repair that are potential targets of the APC/C. Firstly, proteins were identified that contain evolutionary conserved and sterically accessibly APC/C targeting domains (KEN and D-boxes) using bioinformatics. Within this group of proteins with a known role in DNA repair were highlighted. Subsequently, a quantitative mass-spec approach was used to identify those proteins, whose abundance was readily lost upon mitotic exit. Reassuringly, we could identify many previously identified APC/C targets, that all showed loss of abundance during mitotic exit. We subsequently searched for DNA repair-associated factors that were down-regulated during mitotic exit and among those, we identified multiple topoisomerase subunits as likely APC/C substrates. We could subsequently confirm a role for the APC/C in regulating protein levels of these novel substrates using RNAi-mediated depletion of Cdh1 and live cell microscopy. In conclusion, we here present a novel method to identify APC/C targets and used this approach to uncover novel regulatory aspects of cell cycle-regulated DNA repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2011-2960