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. Author manuscript; available in PMC: 2021 Jul 12.
Published in final edited form as: Cell Rep. 2019 Sep 17;28(12):3167–3181.e6. doi: 10.1016/j.celrep.2019.08.041

Figure 7. Depletion of XPF Prevents the Induction of DSBs upon Depletion of TDP1 in Quiescent Cells.

Figure 7.

(A and B) Quiescent cells were transfected with the indicated siRNAs and co-stained for γH2AX and p53BP1. To compare XPF-proficient (siCtrl) and XPF-deficient (siXPF) cells upon depletion of TDP1 (A) or SETX (B), γH2AX induction was normalized to 1 in both siCtrl and siXPF cells (means ± SEM; n = 3). **p < 0.01, ***p < 0.001 (two-tailed unpaired t test).

(C) Viability of siRNA-transfected quiescent cells treated with 25 μM CPT (means ± SEM of triplicates).

(D) Model for the induction of DSBs during transcription. (a) TOP1 removes both positive and negative DNA supercoiling (Sc) generated during transcription. (b) Stabilization of a TOP1cc on the template strand blocks Pol II transcription and promotes R-loop formation. (c) The transcription-blocking TOP1cc is partially proteolyzed by the ubiquitin-proteasome system, which reveals the SSB. The R-loop is cleaved by a dual incision mediated by XPG and XPF or FEN1 and XPF (legend continued on next page) generating a SSB on the opposing DNA strand. Stalled Pol II is degraded and/or removed from the chromatin. (d) The two DNA strands separate forming a DSB. Boxes indicate human disorders associated with increase of TOP1cc (blue) or R-loop levels (red). Genes mutated in these disorders are indicated. ALS, amyotrophic lateral sclerosis; AOA, ataxia with oculomotor apraxia; AT, ataxia telangiectasia; SCAN, spinocerebellar ataxia with axonal neuropathy; SMA, spinal muscular atrophy; SMN, survival motor neuron.

See also Figure S7.