A ubiquitin-proteasome pathway for the repair of topoisomerase I-DNA covalent complexes.
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.
Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Chao-Po
CP
Lin
Yi
Y
Ban
Yi Lisa
YL
Lyu
Shyamal D
SD
Desai
Leroy F
LF
Liu
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D020552:Comet Assay; D004249:DNA Damage; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D004264:DNA Topoisomerases, Type I