A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes

Chao-Po Lin,Yi Ban,Yi Lisa Lyu,Shyamal D Desai,Leroy F Liu

doi:10.1074/jbc.m803493200

Abstract

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 (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. 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. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. 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 through a PARP1-dependent process.

Highlights

Studies using camptothecins (CPTs) have generated a wealth of information on the structure and biology of Top1 cleavage complexes
It has been hypothesized that reversible Top1 cleavage complexes are processed into DNA damage through their interactions with cellular machineries associated with active DNA replication and transcription [7,8,9,10,11]
CPT Induces Distinct DNA Damage Signals Depending on the Growth State of WI-38 Cells—To study the roles of DNA replication and transcription in processing Top1-DNA cleavage complexes, CPT-induced DNA damage signals were measured in both proliferating and nonproliferating primary human lung fibroblasts, WI-38 cells

Summary

Introduction

Studies using camptothecins (CPTs) have generated a wealth of information on the structure and biology of Top1 cleavage complexes (reviewed in Refs. 3 and 5). Like Top1 down-regulation, CPT-induced SSBs and ATM autophosphorylation were shown to be transcription- and proteasome-dependent, consistent with the notion that transcription and proteasome are required for the processing of Top1 cleavage complexes into DNA damage.

Results

Conclusion