Proteasome-dependent Processing of Topoisomerase I-DNA Adducts into DNA Double Strand Breaks at Arrested Replication Forks

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 replicationdependent 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. ␥-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 ␥-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.Eukaryotic DNA topoisomerase I (Top1) 3 catalyzes the breakage/reunion of DNA by transiently nicking one strand of the DNA duplex through the formation of a reversible Top1-DNA covalent complex (reviewed in Refs.

Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage

“…Next, we investigated the involvement of TIP and RIP pathways in the activation of CPT-induced DDR and found that RPA phosphorylation was mainly initiated through the RIP pathway of TOP1cc, as previously reported [21]. The involvement of RIP pathway in the activation of CPT-induced DDR is supported by a recent study showing the role of TOP1cc-mediated and replication-dependent DNA DSBs in the CPT-induced γ-H2AX activation [12]. Nevertheless, several DDR activations, including γ-H2AX, ATM and Chk2 phosphorylation, have also been observed in nonreplicating, post-mitotic cells treated with CPT [11], suggesting a replication-independent mechanism for the activation of CPT-induced DDR.…”

“…CPT treatment has been shown to induce the formation of TOP1cc and subsequent activation of signaling molecules and corresponding DDR [11][12][13]. We first used the alkaline comet assay to detect DNA SSBs induced by CPT treatment and to characterize the reversibility of these breaks.…”

“…The UPP pathway and Tdp1 have been shown to be involved in the removal of TOP1 from enzyme-linked DNA breaks. Owing to the ability in removal of enzyme from cleavable complex, the UPP pathway and Tdp1 have thus been suggested to participate in the repair of TOP1-and/or TOP2-linked DNA breaks [6,[12][13][14][15]. In addition, the CPT-induced degradation of TOP1 (namely TOP1 downregulation) is initiated by the collision between RNA polymerase and TOP1cc, followed by proteolysis through the UPP pathway, like those observed in the process of TOP2cc downregulation [4,14,22,23].…”

“…We have further shown that these two pathways play distinctive roles in the activation of etoposide-induced DDR and signaling [14]. Notably, cellular activities in the TOP2cc-processing pathways have also been shown to be involved in converting TOP1cc into different forms of DNA lesions [12,13,[17][18][19][20][21]. Different from that of TOP2cc, TOP1 is linked covalently to the 3′ terminus of DNA single-stranded break (SSB) in TOP1cc.…”

“…The structural information [8][9][10] and recent studies on the roles of cellular activities and pathways for TOPcc-induced DDR [11][12][13][14][15][16] have suggested that the interactions between TOPcc and cellular activities, such as proteasomal degradation, might process DNA break embedded in the enzyme center into more detectable DNA lesions. These DNA lesions are differentially detected and then followed by the activation of various DDR.…”