Proteasome-dependent processing of topoisomerase I-DNA adducts into DNA double strand breaks at arrested replication forks

Chao Po Lin, Yi Ban, Yi Lisa Lyu, Leroy-Fong Liu

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (CPTs) (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged, UV-irradiated, or alkylated DNA). It has been proposed that Top1 cleavage complexes arrest advancing replication forks, triggering the formation of DNA double strand breaks (DSBs) because of replication fork runoff at the Top1 cleavage complex sites on the leading strand. In this study, we show that the formation of replication-dependent DSBs requires the ubiquitin-proteasome pathway in CPT-treated cells. First, the proteasome inhibitor MG-132 specifically inhibited CPT-induced 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. γ-H2AX, phosphorylated ataxia telangiectasia mutated (Ser-1981), and phosphorylated Chk2 (Ser-33/35)) that are characteristic for DSBs. Knocking down the 20 S proteasome maturation protein also supported the requirement of the proteasome activity for CPT-induced DSBs. Second, CPT-induced DSB signals were shown to require ubiquitin, ubiquitinactivating enzyme (E1), a CUL-3-based ubiquitin ligase (E3), and the formation of Lys-48-linked polyubiquitin chains on Top1. Third, immunocytochemical studies revealed that the CPT-induced formation of γ-H2AX foci occurred at the replication forks and was attenuated by co-treatment with the proteasome inhibitor 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.

Original languageEnglish
Pages (from-to)28084-28092
Number of pages9
JournalJournal of Biological Chemistry
Volume284
Issue number41
DOIs
Publication statusPublished - Oct 9 2009
Externally publishedYes

Fingerprint

Camptothecin
Type I DNA Topoisomerase
Double-Stranded DNA Breaks
DNA Adducts
Proteasome Endopeptidase Complex
DNA
Processing
irinotecan
Proteasome Inhibitors
Ubiquitin
Runoff
Polyubiquitin
Topotecan
Ataxia Telangiectasia
DNA Cleavage
Comet Assay
Ubiquitin-Protein Ligases
Hydroxyurea
Ionizing radiation
Ionizing Radiation

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Proteasome-dependent processing of topoisomerase I-DNA adducts into DNA double strand breaks at arrested replication forks. / Lin, Chao Po; Ban, Yi; Lyu, Yi Lisa; Liu, Leroy-Fong.

In: Journal of Biological Chemistry, Vol. 284, No. 41, 09.10.2009, p. 28084-28092.

Research output: Contribution to journalArticle

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