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. 2014 Dec 18;56(6):777-85.
doi: 10.1016/j.molcel.2014.10.020. Epub 2014 Nov 26.

Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability

Julie Sollier  1 , Caroline Townsend Stork  1 , María L García-Rubio  2 , Renee D Paulsen  1 , Andrés Aguilera  2 , Karlene A Cimprich  3
Affiliations

Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability

Julie Sollier et al. Mol Cell. .

Abstract

R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

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Figures

Figure 1
Figure 1. AQR knockdown leads to DSBs formation and R-loop accumulation
(A) P-KAP1, P-CHK1 and pS33-RPA2 levels in HeLa cells transfected with siLUC and two siAQR for 72 hours. (B) Neutral comet assay in HeLa cells transfected with siLUC or siAQR. Scale bar represents 50 μm. (C) Quantification of comet tail moment for the experiment described in (B). a.u. = arbitrary units. ****p<0.0001. (D) Quantification of RNA-DNA hybrids detected by slot-blot with S9.6 antibody in HeLa cells, with fold enrichment relative to siLUC signal. Errors bars are SEM of three biological replicates. (*p<0.05 by student's t-test). (E) Immunostaining with S9.6 (red) and nucleolin (green) antibodies in HeLa cells transfected with siRNA and fixed after 48 hours. The nucleus (stained with Hoechst) is outlined. Scale bar represents 10 μm. The levels of all panels were adjusted equally in Adobe Photoshop. (F) Quantification of S9.6 signal per nucleus after nucleolar removal for the experiment described in (E), shown as box and whiskers plot. ****p<0.0001. See also Figure S1.
Figure 2
Figure 2. R-loop-induced DNA damage depends on XPF and XPG
(A) P-KAP1 level in HeLa cells transfected with siXPG or siLUC 24 hours prior to transfection with siLUC or siAQR. (B) Neutral comet assay in HeLa cells treated as in Figure 2A. Scale bar represents 50 μm. (C) Quantification of comet tail moment for the experiment described in (B). a.u. arbitrary units. ****p<0.0001. (D, E) P-KAP1 level in XPG- and XPF-patient cell lines either complemented or not with the corresponding wild-type proteins, and transfected with siLUC, siAQR#2 or siAQR#3. (F, G) Quantification of percent γH2AX-positive cells in XPG- and XPF-patient cell lines either complemented or not with the wild-type or nuclease-dead proteins, and transfected with indicated siRNA. (SEM, n=3). (H) Immunostaining with S9.6 (red) and nucleolin (green) antibodies in HeLa cells transfected with siXPG or siLUC 24 hours before transfection with siLUC or siAQR. A merge of the two channels is shown, with the nucleus (stained with Hoechst) outlined. Scale bar represents 10 μm. The levels of all panels were adjusted equally in Adobe Photoshop. (I) Quantification of S9.6 immunofluorescence intensity per nucleus for the experiment described in (H), shown as box and whiskers plot. ****p<0.0001.
Figure 3
Figure 3. The processing of R-loops by the endonucleases XPF and XPG is a general and conserved mechanism
(A, B). P-KAP1 level in HeLa cells transfected with siXPG or siLUC 24 hours before transfection with siASF or siSETX. (C) γH2AX intensity in HeLa-TetON-RNase H1 cells transfected with siLUC or siXPG for 48 hours and treated for 2 hours with 5μM camptothecin. Doxycycline (500 ng/μl) was added in combination with siRNAs where indicated. a.u. = arbitrary units. (D) Frequencies of recombination in the LY direct-repeat system. Each data represents the median of 3-4 independent experiments. Error bars represent the standard error of the median (SEM, n=3-4). See also Figure S2 and Table S1.
Figure 4
Figure 4. R-loop processing requires TC-NER factors but not XPC
(A) P-KAP1 level in HeLa cells transfected with siXPA or siLUC 24 hours before transfection with siLUC or siAQR. (B) P-KAP1 level in HeLa cells transfected with siXPB, siXPD or siLUC 24 hours before transfection with siLUC or siAQR. (C) P-KAP1 level in HeLa cells transfected with siXPC or siLUC 24 hours before transfection with siLUC or siAQR. (D) P-KAP1 level in HeLa cells transfected with siCSB or siLUC 24 hours before transfection with siLUC or siAQR. (E) Model for how an R-loop is processed into a DSB. The stalling of the RNA polymerase allows CSB to recruit the endonucleases XPF and XPG. XPF and XPG generate a gap that can be converted into a DSB through DNA replication and/or XPF and XPG cleave the R-loop on both strands producing a DSB. See also Figure S3.
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