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National Yang-Ming University
Institute of Genetics
Thesis Advisor: Leroy F. Liu, PhD
Graduate Program of Cellular and Molecular Pharmacology
Wednesday, October 7, 2009
Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs. Top1 cleavage complexes collide with transcription and replication machineries and trigger the degradation of Top1, a phenomenon termed Top1 down-regulation. We have investigated the role of Top1 down-regulation in the formation of camptothecin (CPT)-induced single-strand breaks (SSBs) and double strand breaks (DSBs). First, studies using quiescent (serum-starved) human WI-38 cells demonstrated that CPT was shown to induce Top1 down-regulation, which paralleled the induction of DNA SSBs and ATM autophosphorylation, all of which were abolished by the proteasome inhibitor, MG-132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair.
Second, we show that the formation of CPT-induced, replication-dependent DSBs requires the ubiquitin-proteasome pathway. The proteasome inhibitor, MG-132, specifically inhibited CPT-, but not ionizing radiation- or hydroxyurea-, induced DSBs as revealed by both the neutral comet assay and measurements of the specific DNA damage signals (e.g. -H2AX, phosphorylated ATM (Ser-1981) and Chk2 (Ser-33/35)) that are characteristic for DSBs. Knocking down the 20S proteasome maturation protein (POMP) also supported the requirement of the proteasome activity for CPT-induced DSBs. Consistently, CPT-induced DSB signals were also shown to require ubiquitin, ubiquitin E1, a CUL-3-based E3 ligase, and the formation of Lys-48-linked polyubiquitin chains on Top1. Finally, immunocytochemical studies revealed that the CPT-induced formation of gamma-H2AX foci occurred at the replication forks and was attenuated by co-treatment with MG-132. In the aggregate, these results support a replication fork collision model in which Top1 cleavage complexes at the arrested replication forks are degraded by proteasome prior to replication fork runoff on the leading strand to generate DSBs.