Topoisomerase I poisoning results in PARP-mediated replication fork reversal

Abstract

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics.

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

“…The frequency of reversed forks in untreated PARG-depleted cells is close to that observed with mild CPT treatments in control HeLa cells (36%) (Fig. 3B) and that reported upon Top1 poisoning in yeast, Xenopus egg extracts, U2OS cells, and mouse embryonic fibroblasts (15 to 40%) (11). Furthermore, PARG depletion increased the frequency of reversed forks upon CPT treatments only marginally (from 36% to 42%) (Fig.…”

“…Overall, the effects of PARG depletion on EdU incorporation and H2AX phosphorylation were comparable to those induced by mild treatment (25 nM) with the prototypical Top1 poison camptothecin (CPT) (Fig. 1D and E), previously shown to interfere with replication fork progression and to induce fork reversal in the absence of detectable chromosomal breakage (11). Indeed, PAR accumulation-which is cell cycle independent upon H 2 O 2 treatment-occurred specifically in S-phase (EdUpositive [EdU ϩ ]) cells upon PARG depletion (Fig.…”

“…DSB detection by pulsed-field gel electrophoresis (PFGE) was performed as reported previously (11). Briefly, cells were embedded in a 0.8% agarose plug (2.5 ϫ 10 5 cells/plug), digested in lysis buffer (100 mM EDTA, 1% [wt/vol] sodium lauryl sarcosine, 0.2% [wt/vol] sodium deoxycholate, 1 mg/ml proteinase K) at 37°C for 48 h, and washed in 10 mM Tris-HCl (pH 8.0)-100 mM EDTA.…”

“…The reason behind the sensitivity of HR-deficient cells to PARP inhibition is thought to be the accumulation of single-stranded DNA (ssDNA) breaks in the absence of PAR synthesis, leading to replication fork collapse and double-stranded breaks (DSBs), which would then require HR factors for repair (8,10). Recently, PARP activity has also been reported to play a role in the control of replication fork reversal upon topoisomerase 1 poisoning (11). Upon auto-PARylation, PARP1 interacts with the RecQ1 helicase and inhibits its specific fork restart activity, thereby transiently preventing restart of the reversed forks until repair of the damage has occurred (12).…”

Replication stress and cancer: It takes two to tango

“…The main role proposed for PARylation in genome stability is linked to the recruitment of specific proteins through PAR binding domains such as the macro domain. At replication forks, the establishment of a PAR scaffold is necessary for the reversal of forks generated by TopI inhibition [73,74]. In addition, PAR protects reversed forks from MRE11 mediated degradation by either physically impeding the access to the fork or by cooperating in the recruitment of BRCA2 and RAD51.…”