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

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 downregulation, 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.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. 1-4). It has been well established that anticancer camptothecins (e.g. irinotecan and topotecan) as well as various structurally perturbed DNAs (e.g. UV adducts, abasic sites, base mismatches, uracil incorporation, nicks and gaps, and oxidized DNA lesions) stabilize reversible Top1-DNA covalent complexes, often referred to as Top1 cleavable or cleavage complexes (reviewed in Refs. 3 and 5).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). It is well established that CPTs exert their antitumor activity through their specific stabilization of Top1 cleavage complexes (6). Top1 cleavage complexes are unique DNA lesions, characterized by their reversibility and Top1-concealed single-strand breaks (SSBs) (6). 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 (7-11).The role of active DNA replication in the processing of Top1 cleavage complexes into DNA damage was initially sugges...

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

“…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.…”

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

“…[12][13][14] Chlorambucil readily forms mono-adducts (as well as some crosslinks) with dsDNA, 15 whereas camptothecin does not react with the DNA strand directly, but rather interfere with topoisomerase I activity by blocking the re-sealing of the DNA, which may eventually result in frank strand breaks. 16 Surprisingly, we find that sequence-targeted PNAs that can react covalently with the DNA helix exhibit a highly inhibitory effect on gene correction in nuclear extract and in a cell culture, extra-chromosomal assay. In contrast, a conventional triplex forming PNA (TFP) showed little or no effect on gene correction, whereas a PNA-camptothecin conjugate was found to stimulate gene correction events in nuclear extracts, but not in a cell culture assay.…”

“…Permanently stalled topo I/DNA complexes are tagged with ubiquitin for proteasomal degradation. 16 Thus, an increased concentration of ubiquitin could increase the rate of protein/DNA complex processing, thereby increasing the gene correction rate. Also, ubiquitin serves as modulator of several DNA repair mechanisms and consequently, increased concentrations of ubiquitin could increase the processing rates for these reactions.…”

“…Such complexes are not tolerated, and are subject to cellular protease degradation, which can lead to the formation of DNA single strand breaks. 16 Previously, TFO-camptothecin conjugates have been shown to promote DNA strand break in vitro. 25 PNA oligomers were incubated with the vector, photo-activated (UVA irradiation) when necessary, and mixed with the appropriate DNA donor before incubation in mammalian cell nuclear extract or co-transfection into mammalian cells.…”