TDP1 suppresses chromosomal translocations and cell death induced by abortive TOP1 activity during gene transcription

Abstract

DNA topoisomerase I (TOP1) removes torsional stress by transiently cutting one DNA strand. Such cuts are rejoined by TOP1 but can occasionally become abortive generating permanent protein-linked single strand breaks (SSBs). The repair of these breaks is initiated by tyrosyl-DNA phosphodiesterase 1 (TDP1), a conserved enzyme that unlinks the TOP1 peptide from the DNA break. Additionally, some of these SSBs can result in double strand breaks (DSBs) either during replication or by a poorly understood transcription-associated process. In this study, we identify these DSBs as a source of genome rearrangements, which are suppressed by TDP1. Intriguingly, we also provide a mechanistic explanation for the formation of chromosomal translocations unveiling an error-prone pathway that relies on the MRN complex and canonical non-homologous end-joining. Collectively, these data highlight the threat posed by TOP1-induced DSBs during transcription and demonstrate the importance of TDP1-dependent end-joining in protecting both gene transcription and genome stability.

SCAN1 mutant TDP1 blocks the repair of DSB induced by TOP1 activity during gene transcription and promotes genome reorganisations and cell death in quiescent cells

“…We previously reported that TDP1 is required for the repair of TOP1-induced DSBs in quiescent hTERT-immortalized retinal pigment epithelial cells (RPE-1) cells(19). To evaluate possible TOP1-induced DSB repair defects in TDP1-associated neurological disease SCAN1, we conducted complementation experiments on TDP1 -/- RPE-1 cells with an empty vector (EV), a wild-type TDP1 or the SCAN1-associated mutation H493R (hereafter TDP1 H493R ) which were under the control of a doxycycline-inducible promoter (see methods for details).…”

“…1b). We have previously shown that CPT-induced DSBs under these conditions are entirely dependent on gene transcription(19). Importantly, expression of wild-type TDP1 suppressed the accumulation of CPT-induced DSBs to a much greater extent than TDP1 H493R , which remained close to the levels observed with EV.…”

“…Next, we studied the formation of transcription associated chromosomal reorganisations in TDP1 -/- complemented cells upon CPT treatment. Cells were treated and maintained in G0/G1 during 6□h after treatment with CPT and then released for the isolation of metaphases(19) (Figure 5a). However, PNKP-depletion cells did not provoke a significant increase in chromosomal translocations (Fig.…”

“…In this study, we addressed the impact of the H493R mutation in TDP1, which causes the autosomal recessive neurodegenerative disease SCAN1, on the repair of TOP1-induced DSBs in quiescent cells. We previously reported that the loss of TDP1 significantly delays TOP1-induced DSB repair in quiescent RPE-1 cells promoting genome instability and cell death (19). Remarkably, our complementation experiments on TDP1 -/- RPE-1 cells showed that TDP1 H493R overexpression not only fails to suppress this delay but also results in fully defective TOP1-induced DSB repair.…”

“…We recently showed that TDP1-dependent DSB repair suppresses CPT-induced chromosome translocations generated by TOP1 abortive activity demonstrating that TOP1-induced DSB during transcription can result in replication-independent genome reorganisations(19). To see if this is a general characteristic of other proteins in the SSBR pathway we depleted PNKP, the first enzymatic activity after TDP1.…”