XAB2 dynamics during DNA damage- dependent transcription

Donnio, Cerutti etal. eLife 2022;0:e77094. DOI: https://doi.org/10.7554/eLife.77094 1 of 24

XAB2 dynamics during DNA damage-

dependent transcriptioninhibition

Lise- Marie Donnio

†

, Elena Cerutti

†

, Charlene Magnani, Damien Neuillet,

Pierre- Olivier Mari, Giuseppina Giglia- Mari*

Institut NeuroMyogène (INMG), CNRS UMR 5310, INSERM U1217, Université Claude

Bernard Lyon 1, Lyon, France

Abstract Xeroderma Pigmentosum group A- binding protein 2 (XAB2) is a multifunctional protein

playing a critical role in distinct cellular processes including transcription, splicing, DNA repair,

and messenger RNA export. In this study, we demonstrate that XAB2 is involved speciﬁcally and

exclusively in Transcription- Coupled Nucleotide Excision Repair (TC- NER) reactions and solely for

RNA polymerase 2 (RNAP2)- transcribed genes. Surprisingly, contrary to all the other NER proteins

studied so far, XAB2 does not accumulate on the local UV- C damage; on the contrary, it becomes

more mobile after damage induction. XAB2 mobility is restored when DNA repair reactions are

completed. By scrutinizing from which cellular complex/partner/structure XAB2 is released, we have

identiﬁed that XAB2 is detached after DNA damage induction from DNA:RNA hybrids, commonly

known as R- loops, and from the CSA and XPG proteins. This release contributes to the DNA

damage recognition step during TC- NER, as in the absence of XAB2, RNAP2 is blocked longer on

UV lesions. Moreover, we also demonstrate that XAB2 has a role in retaining RNAP2 on its substrate

without any DNA damage.

Editor's evaluation

This manuscript will be of interest for individuals working in genome stability, speciﬁcally on the

repair of UV damage and nucleotide excision repair (NER). The authors report that the transcription-

coupled NER factor XAB2 is mobilized after DNA damage and that XAB2 keeps RNA Pol2 engaged

on chromatin. XAB2 mobilization appears to be caused by transcription blockage imposed by the

DNA damage.

Introduction

The DNA molecule in our cells’ nucleus forms the instruction manual for proper cellular functioning.

Unfortunately, the integrity of our DNA is continuously challenged by a variety of endogenous and

exogenous agents (e.g., ultraviolet light [UV], cigarette smoke, environmental pollution, oxidative

damage, etc.). These DNA lesions interfere with DNA replication, transcription, and cell cycle progres-

sion, leading to mutations and cell death, which may cause cancer, inherited diseases, or aging (Chat-

terjee and Walker, 2017).

To prevent the deleterious consequences of persisting DNA lesions, all organisms are equipped

with a network of efﬁcient DNA repair systems. One of these systems is the Nucleotide Excision

Repair (NER) which removes helix- distorting DNA adducts caused by UV such as Cyclo- Pyrimidine

Dimers (CPDs) and 6- 4 Photoproducts (6- 4PPs) (Giglia- Mari etal., 2011).

In mammals, the different steps of NER require more than 30 different proteins that are recruited

sequentially to the DNA damage site, as demonstrated by the different studies of NER proteins

kinetics (Moné etal., 2004; Politi etal., 2005; Rademakers etal., 2003; van den Boom etal., 2004;

RESEARCH ARTICLE

*For correspondence:

[email protected]

†

These authors contributed

equally to this work

Competing interest: The authors

declare that no competing

interests exist.

Funding: See page 22

Preprinted: 24 December 2021

Received: 25 January 2022

Accepted: 25 July 2022

Published: 26 July 2022

Reviewing Editor: Wolf- Dietrich

Heyer, University of California,

Davis, United States

etal. This article is distributed

under the terms of the Creative

Commons Attribution License,

which permits unrestricted use

and redistribution provided that

the original author and source

are credited.

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Zotter etal., 2006). The ﬁrst step of NER consists of damage recognition, followed by the opening

of the DNA duplex, dual incisions on both sides of the damage, excision of 24–32 oligonucleotides

containing the damage and, ﬁnally, gap- ﬁlling by repair DNA synthesis.

NER is divided into two subpathways depending on DNA lesions position within the genome.

The Global Genome repair (GG- NER) will detect and repair lesions throughout the genome, whereas

Transcription- Coupled repair (TC- NER) is associated with RNA polymerase 2 (RNAP2) to repair lesions

on the transcribed strand of active genes (Marteijn etal., 2014).

The NER system has been linked to rare human diseases, classically grouped into three distinct

NER- related syndromes. These include the highly cancer- prone disorder Xeroderma Pigmentosum

(XP) and the two progeroid diseases: Cockayne Syndrome (CS) and Trichothiodystrophy (TTD). Impor-

tantly, CS and TTD patients are not cancer prone but present severe neurological and developmental

features (Hanawalt, 1994).

Xeroderma Pigmentosum group A (XPA)- binding protein 2 (XAB2) is a highly conserved protein

of 100kDa and consists of 15 tetratricopeptide repeat (TPR) motifs that carry out protein–protein

interactions. XAB2 protein was identiﬁed as a protein interacting with XPA, a NER factor, using a

yeast two- hybrid system (Nakatsu etal., 2000). Next, it has been shown that XAB2 interacts also

with the TC- NER- speciﬁc factors, CSA and CSB, and the elongating form of RNAP2 (Nakatsu etal.,

2000). XAB2 is also essential for early mouse embryogenesis, as demonstrated by the preimplantation

lethality observed in XAB2 knockout mice (Yonemasu etal., 2005).

Downregulation of XAB2, using either anti- XAB2 or siRNA, inhibits normal RNA synthesis and the

recovery of RNA synthesis after UV irradiation (Kuraoka etal., 2008; Nakatsu etal., 2000). Further-

more, injection of anti- XAB2 in GG- NER- deﬁcient cells signiﬁcantly reduces UV- induced Unscheduled

DNA Synthesis (UDS) during repair (Nakatsu etal., 2000). These results suggest the involvement of

XAB2 in transcription and TC- NER.

Further studies have shown that XAB2 is a component of the Prp19/XAB2 complex (Aquarius [AQR],

XAB2, Prp19, CCDC16, hISY1, and PPIE) or Prp19/CDC5L- related complex required for pre- mRNA

splicing (Kuraoka et al., 2008). XAB2, as well as PRP19 and AQR, has been involved in the DNA

damage response (DDR) (Maréchal etal., 2014; Onyango etal., 2016; Sakasai etal., 2017). Indeed,

XAB2 is essential for homologous recombination (HR) by promoting the end resection step (Onyango

etal., 2016). PRP19 is a sensor of RPA- ssDNA after DNA damage (Maréchal etal., 2014) and AQR

contributes to the maintenance of genomic stability via regulation of HR (Sakasai etal., 2017).

Interestingly, AQR has a role in removing R- loops, a three- stranded nucleic acids structure

composed of a DNA:RNA hybrid and the associated nontemplate single- stranded DNA (Sollier etal.,

2014). These structures can form during transcription, when an RNA molecule emerging from the

transcription machinery hybridizes with its DNA template. They are found abundantly in human gene

promoters and terminators where RNA processing occurs (Wang etal., 2018).

Despite the knowledge acquired in the last decades on XAB2 and its different cellular roles, little

is known about the exact crosstalk and dynamics between its diverse cellular functions, speciﬁcally

between DNA repair transcription and splicing. In this work, we describe the molecular dynamics of

XAB2 within the cell after UV- damage induction and during the TC- NER repair process. We determined

in vivo that, in the absence of XAB2, Transcription- Coupled repair reactions are impaired, consequently,

restart of transcription after UV damage is abolished. Surprisingly, unlike all the other NER proteins

studied so far, the mobility of XAB2 is increased after irradiation, and XAB2 shows no accumulation

on local UV lesions. This changing dynamic is not restored until DNA repair is completed. Indeed, in

damaged TC- NER- deﬁcient cells, XAB2 remains more mobile. Interestingly, we demonstrate that, after

DNA damage induction, XAB2 is not released from the splicing complex but is detached from R- loops,

a recently XAB2 identiﬁed substrate (Goulielmaki etal., 2021). Additionally, we investigate the rela-

tion between XAB2 and RNAP2, demonstrating that XAB2 retains RNAP2 on its substrate. Moreover, in

the absence of XAB2, RNAP2 interacts strongly and durably with both types of UV lesions (6- 4PPs and

CPDs), suggesting a role of XAB2 in the DNA damage recognition step of TC- NER.

Results

XAB2 is involved in TC-NER process

Two decades ago, Tanaka’s research group demonstrated the involvement of XAB2 in the NER

pathway (Kuraoka etal., 2008; Nakatsu etal., 2000). However, the dynamics of XAB2 during the

Research article

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DNA repair process remained to be elucidated. We aimed to study the shuttling between its different

functions when DNA damage is induced. Firstly, we wanted to verify that XAB2 is exclusively involved

in TC- NER reactions.

The well- known standard assay used to quantify NER activity is the UDS, which measures repli-

cation activity outside the S- phase after ultraviolet light (UV- C) treatment. This technique quantiﬁes

the reﬁlling of the single- strand DNA gap by the DNA replicative machinery. When we performed

an Unschelduled DNA synthesis (UDS) assay in XAB2- silenced cells, no decreased level of UDS

was observed (as well as in mock- treated cells; Figure1A blue and black columns, Figure1B and

Figure1—ﬁgure supplement 1). As a positive control, when we silenced the excision repair factor

XPF, we observed a strong reduction in UDS level (Figure1A, red column, Figure1B, and Figure1—

ﬁgure supplement 1 ). This result shows that XAB2 is not involved in the GG- NER subpathway, but

does not exclude an involvement of XAB2 in the TC- NER subpathway.

The commonly used assay measuring TC- NER activity is the RNA Recovery Synthesis (RRS). This

assay measures the newly transcribed RNA by incorporating a nucleoside analog coupled to a ﬂuoro-

phore. The experiment is conducted at different time points after UV irradiation (0, 3, 16, and 24hr)

in order to quantify the decline in transcriptional activity (3hr after UV damage) and the restart of

transcriptional activity (16–24hr after UV irradiation). In XAB2- silenced cells, no restart of transcrip-

tion after UV damage was observed (Figure1C, blue column and Figure1—ﬁgure supplement 2A),

as well as in siXPF- treated cells due to the inability to repair DNA lesions (Figure1C, red column

and Figure2—ﬁgure supplement 2A) and in contrast with siMock- treated cells (Figure1C, black

column and Figure1—ﬁgure supplement 2A). In XAB2- silenced cells and in the absence of DNA

damage, we observed a decreased level of nascent RNA synthesis when the nucleoside analog EU

was incubated for 1 hr, accordingly to the result of Tanaka’s group (Figure1—ﬁgure supplement 2B;

Kuraoka etal., 2008; Nakatsu etal., 2000). However, when EU is incubated for 2 hr (time point used

for RRS assay) we observed an increase of nascent RNA in XAB2- silenced cells compared to control

cells (Figure1—ﬁgure supplement 2B). As expected, silencing XPF protein does not affect basal

transcription (Figure1—ﬁgure supplement 2C). RRS results demonstrated an involvement of XAB2

in the TC- NER subpathway but did not discriminate between a role in the repair reaction per se or in

the Restart of Transcription after Repair (Mourgues etal., 2013).

In order to discriminate this point, we performed an assay designed previously in our group that

precisely measures repair replication during TC- NER: the TCR- UDS assay (Mourgues etal., 2013).

For this assay, we performed the UDS assay in GG- NER- deﬁcient cells using XPC (Xeroderma Pigmen-

tosum complementation group C) mutant cells (XP4PA- SV). The cells were transfected with speciﬁc

siRNAs and then locally irradiated with UV- C through a ﬁlter. In order to precisely localize DNA-

damaged areas, a γH2AX coimmunoﬂuorescence labeling was performed, and repair replication was

quantiﬁed. In siXPF- treated XPC- negative cells, both the GG- NER and the TC- NER pathways are

compromised and, as expected, low TCR- UDS levels were observed compared to siMock- treated

cells (Figure1D, red and black columns and Figure 1—ﬁgure supplement 3). Silencing of XAB2

results in a decrease in TCR- UDS levels (Figure1D, blue column and Figure1—ﬁgure supplement 3).

This result demonstrates a role of XAB2 in the repair reaction itself, its silencing preventing the DNA

synthesis associated with the excision of UV lesions on actively transcribed genes.

Next, we decided to investigate by PLA (Proximity Ligation Assay) whether XAB2 can interact with

6- 4PPs and CPDs lesion, helix- distorting DNA adducts caused by UV. We observed a strong interac-

tion between XAB2 and 6- 4PPs 1hr after 10J/m² irradiation (Figure2A, C). This interaction correlates

with the amount of 6- 4PPs and, as expected, decreases during repair (Figure 2B, C), while XAB2

concentration does not change after irradiation (Figure2B, C). The same result is obtained in PLA

experiment between XAB2 and CPDs (Figure2—ﬁgure supplement 1). These results demonstrate

that XAB2 interacts directly with or is in the proximity of 6- 4PP and CPD lesions until their removal.

Recently, we demonstrate that a fully functional NER mechanism is necessary for the repair of ribo-

somal DNA (rDNA), genes transcribed by the RNA polymerase 1 (RNAP1) (Daniel etal., 2018). To

investigate the involvement of XAB2 in the repair of ribosomal genes, the level of RNAP1 transcription

was measured at different time points after UV irradiation by using a speciﬁc ribosomal RNA probe

coupled to a ﬂuorophore, as described previously (Daniel etal., 2018). This probe recognizes the 5′

end of the rDNA transcript, the 47S pre- rRNA (upstream from the ﬁrst site cleaved rapidly during rRNA

processing) (a sketch of the 47S is depicted in Figure2—ﬁgure supplement 2A). In siMock- treated

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cells, we observed a decrease of 47S levels 3hr after UV- C exposure and the restart of RNAP1 tran-

scription 40hr after irradiation (Figure2—ﬁgure supplement 2B, black column and Figure2—ﬁgure

supplement 2D). siCSB- treated cells, deﬁcient for TC- NER, presented a low level of rRNA synthesis

even 40hr after UV- C exposure (Figure2—ﬁgure supplement 2B, violet column, Figure2—ﬁgure

supplement 2C,D). In the absence of XAB2, 40hr after irradiation, the level of 47S returns to the level

of non- irradiated condition, supporting a restart of RNAP1 transcription (Figure2—ﬁgure supple-

ment 2B, blue column and Figure2—ﬁgure supplement 2D).

Figure 1. XAB2 is involved in DNA repair. (A) Quantication of Unscheduled DNA Synthesis (UDS) assay determined by EdU incorporation after local

damage (LD) induction with UV- C (100J/m

) in WT cells (MRC5 cells) treated with siRNAs against indicated factors. Error bars represent the standard

error of the mean (SEM) obtained from at least 30 LDs. (B) Western blot on whole- cell extracts of MRC5 cells treated with siRNA against indicated

factors. (C) Quantication of RNA Recovery Synthesis (RRS) assay determined by EU incorporation after UV- C (10J/m

) exposure in WT cells treated with

siRNAs against indicated factors. Error bars represent the SEM obtained from at least 50 cells. (D) Quantication of TCR- UDS assay determined by EdU

incorporation after LD induction with UV- C (100J/m

) in GG- NER- decient cells (XPC−/− cells) treated with siRNAs against indicated factors. Error bars

represent the SEM obtained from at least 15 LDs. For all graphs, p- value of Student’s test compared to siMock condition: ***<0.001.

The online version of this article includes the following source data and gure supplement(s) for gure 1:

Source data 1. Source data for Figure1A: quantication of UDS siXAB2.

Source data 2. Source data for Figure1C: quantication of RRS siXAB2.

Source data 3. Source data for Figure1D: quantication of TCR- UDS siXAB2.

Source data 4. Figures with the uncropped blots and relevant bands clearly labeled for Figure1B: Western blot siXAB2 efciency.

Source data 5. The original les of the full raw unedited gels for Figure1B: Western blot siXAB2 efciency.

Figure supplement 1. UDS siXAB2.

Figure supplement 2. RRS siXAB2.

Figure supplement 2—source data 1. Source data for Figure1—ﬁgure supplement 2B, C: quantication of RNA synthesis.

Figure supplement 3. TCR- UDS siXAB2.

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Figure 2. XAB2 interacts with the UV lesion 6- 4 Photoproduct (6- 4PP). Quantication of uorescent signal in

the nucleus against the couple XAB2_6- 4PP from Proximity Ligation Assay (PLA) experiment (A) or from the

immunouorescence (IF) done in parallel to PLA assay with the same antibodies dilutions (B). Error bars represent

the standard error of the mean (SEM) obtained from at least 80 cells. P- value of Student’s test compared to No UV

Figure 2 continued on next page

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All these results demonstrate that XAB2 has a function in TC- NER repair reactions speciﬁcally and

exclusively for RNAP2- transcribed genes.

XAB2-splicing complex is released from the DNA damage area

XAB2 is included in a splicing complex composed of ﬁve other proteins: Aquarius (AQR), PRP19,

CCDC16, PPIE, and ISY1 (Kuraoka etal., 2008). In order to explore how the XAB2- splicing complex

behaves after local damage induction, the localization of XAB2, AQR, PRP19, and CCDC16 was

revealed by immunoﬂuorescence assays at different time points after local UV irradiation of the cells.

In this assay, the ﬂuorescence signal from each protein in the damaged area (visualized by a costaining

of γH2AX) was compared to the signal from the rest of the nucleus. Unexpectedly, in contrast with all

other NER proteins studied so far, we observed a relatively rapid (1hr after UV irradiation) release of

XAB2, AQR, PRP19, and CCDC16 from the damaged area (Figure3A, B). The proper localization of

the XAB2- splicing complex is re- established after the completion of DNA repair reactions when the

transcription is fully restarted (16hr after irradiation; Figure3B).

We thus veriﬁed if the entire XAB2- splicing complex was involved in TC- NER or whether only XAB2

played a role in this process. In order to measure the repair capacity of cells silenced for XAB2- related

proteins, we performed UDS, TCR- UDS, and RRS experiments in AQR/PRP19/CCDC16/PPIE/ISY1-

siRNAs treated cells (Figure3—ﬁgure supplements 1–3) and compared the results with XPF- siRNAs

treated cell lines. Our results clearly show that none of the cells silenced for XAB2- related proteins

are deﬁcient in DNA Repair. Moreover, both GG- NER (Figure3—ﬁgure supplement 1) and TC- NER

(Figure3—ﬁgure supplements 2 and 3) are proﬁcient in the absence of AQR, PRP19, CCDC16, PPIE,

or ISY1.

In order to investigate whether XAB2 release from damaged areas was dependent on the TC- NER

reaction, the localization of XAB2 was detected and quantiﬁed within locally damaged areas in

TC- NER- deﬁcient cells: CSA (CS3BE) and CSB (CS1AN) mutant cells. Interestingly, the absence of

CSA and CSB did not hinder the release of XAB2 from locally damaged areas. However, this release

persisted 16hr after UV- C exposure (Figure3C blue and red curves compared to black curve and

Figure3—ﬁgure supplement 4), suggesting that the re- establishment of the proper localization of

XAB2 within the nucleus after the DNA repair process depends either on the repair process per se or

on the restart of transcription after the achievement of DNA repair reactions.

XAB2 dynamic during TC-NER

To further analyze XAB2 mobility within the nuclei, we performed SPOT- FRAP (ﬂuorescent recovery

after photobleaching) experiments. In this technique, ﬂuorescence molecules are photobleached in

a small spot by a high- intensity laser pulse. Subsequently, ﬂuorescence recovery within the bleached

area is monitored over time (Figure 4—ﬁgure supplement 1A). When cells are untreated, the

measure of ﬂuorescence recovery corresponds to the protein intrinsic mobility within the living cells

(Figure4—ﬁgure supplement 1A, black curve). After perturbation of the nuclear environment (e.g.,

condition: ***<0.001. (C) Representative images of the PLA and IF experiments. Nuclei are delimited by dashed

lines. Scale bar: 15µm.

The online version of this article includes the following source data and gure supplement(s) for gure 2:

Source data 1. Source data for Figure2A, B: quantication of PLA and IF XAB2_6- 4PP.

Figure supplement 1. XAB2 interacts with the ultraviolet light (UV) lesions Cyclo- Pyrimidine Dimer (CPD).

Figure supplement 1—source data 1. Source data for Figure2—ﬁgure supplement 1A, B: quantication of

PLA and IF XAB2_CPD.

Figure supplement 2. RNA- FISH siXAB2.

Figure supplement 2—source data 1. Source data for Figure2—ﬁgure supplement 2B: quantication of RNA-

FISH.

Figure supplement 2—source data 2. Figures with the uncropped blots and the relevant bands clearly labeled

for Figure2—ﬁgure supplement 2C: Western blot siCSB efciency.

Figure supplement 2—source data 3. The original les of the full raw unedited gels for Figure2—ﬁgure

supplement 2C: Western blot siCSB efciency.

Figure 2 continued

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Figure 3. Splicing complex is released from DNA damage. (A) Representative confocal images of immunouorescence (IF) against XAB2, AQR, PRP19,

or CCDC16 (green) and γH2AX (red) 1 hr after local damage (LD) induction with UV- C (60J/m

). LDs are indicated by dashed lines. Scale bar: 3µm. (B)

Quantication of the IF signal of the different splicing proteins on the LD after different times of recovery. (C) Quantication of XAB2 signal on LD in

different cell lines after different times of recovery. For both graphs, the signal from the local damage has been subtracted from the background of each

cell. Error bars represent the standard error of the mean (SEM) obtained from at least 20 cells.

The online version of this article includes the following source data and gure supplement(s) for gure 3:

Source data 1. Source data for Figure3B: quantication of splicing complex IF.

Source data 2. Source data for Figure3C: quantication of XAB2 IF.

Figure supplement 1. UDS in splicing complex- silenced cell.

Figure supplement 1—source data 1. Source data for Figure3—ﬁgure supplement 1A: quantication of UDS in splicing complex- silenced cells.

Figure supplement 1—source data 2. Figures with the uncropped blots and relevant bands clearly labeled for Figure3—ﬁgure supplement 1C:

Western blot efciency of siRNA against splicing complex.

Figure supplement 1—source data 3. The orignal les of the full raw unedited gels for for Figure3—ﬁgure supplement 1C: Western blot efciency

of siRNA against splicing complex.

Figure supplement 2. TCR- UDS in splicing complex- silenced cells.

Figure supplement 2—source data 1. Source data for Figure3—ﬁgure supplement 2A: quantication of TCR- UDS in splicing complex- silenced

cells.

Figure supplement 3. RRS in splicing complex- silenced cells.

Figure supplement 3—source data 1. Source data for Figure3—ﬁgure supplement 3A: quantication of RRS in splicing complex- silenced cells.

Figure supplement 4. XAB2 is released from DNA damage also in TC- NER- decient cells.

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DNA damage), a protein can physically interacts with a new substrate or a slower complex, becoming

less mobile (Figure4—ﬁgure supplement 1A, green curve) or on the contrary can be released from

its substrate, becoming more mobile (Figure4—ﬁgure supplement 1A, blue curve). Eventually, the

protein can also have an unchanged mobility (Figure4—ﬁgure supplement 1A, red curve).

We stably transfected a vector expressing a ﬂuorescent version of XAB2 (XAB2- GFP, Figure4—

ﬁgure supplement 1B) in different SV40- immortalized human ﬁbroblast: wild- type cells (MRC5,

Figure4—ﬁgure supplement 1C), CSA- deﬁcient cells (CS3BE) and CSB- deﬁcient cells (CS1AN). In

order to determine the minimum dose of UV- C needed to detect a signiﬁcant difference in XAB2

mobility, MRC5 XAB2- GFP cells were irradiated with doses of UV- C ranging from 2 to 16J/m² and

SPOT- FRAP experiments were performed at different time points following UV irradiation (Figure4A).

Interestingly, we observed a dose- dependent increase in mobility of XAB2 (Figure4A). Doses of UV- C

as weak as 2 and 4J/m² induced a moderate increase in mobility 3 hr post- irradiation and a recovery

of the basal XAB2 mobility within 16 hr post- irradiation (Figure4A, blue and yellow bars). High UV- C

doses (16J/m²) induced a rapid increase in XAB2 mobility (1 hr post- irradiation) and a recovery of

the intrinsic mobility 24 hr post- irradiation (Figure 4A, red bar). At intermediate doses of 8 J/m²

of UV- C, we observed a signiﬁcant increase in XAB2 mobility during repair (3hr after UV- C expo-

sure) and the following return to the normal condition once the repair is completed and transcription

restarted (16hr after irradiation) (Figure4A, green bar). Interestingly, in CSA and CSB mutant cells,

the increase in XAB2 mobility is also observed after 8J/m² irradiation and lasted until 24 hr after

UV- C exposure (Figure4B), witnessing the fact that in these cells, DNA damage is not repaired and

therefore initial intrinsic XAB2 mobility is not restored. Interestingly, without damage, XAB2 mobility

is reduced in TC- NER- deﬁcient cells compared to wild- type cells for still unknown reasons (Figure4B,

black histogram).

The results of these experiments directed us to explore the possibility that the change in XAB2

mobility was due to transcription inhibition and not really to the repair process itself. In order to verify

this hypothesis, XAB2 mobility was measured after DRB (transcription inhibitor) treatment. Surpris-

ingly, results show that XAB2 increased mobility in transcription inhibition conditions is very similar to

the one measured upon UV treatment (Figure4C, red curve compared to blue curve).

As for XAB2, the mobility of the late- stage spliceosomes changes after UV irradiation. This mobi-

lization depends on DDR signaling pathways (Tresini etal., 2015). Key mediators of DDR are the

ATM and ATR kinases, which induce cell cycle arrest and facilitate DNA repair. To demonstrate that

the change in XAB2 mobility is due (or not) to the UV- damage response, we realized the same FRAP

assays in the presence of ATR and ATM inhibitors. Both drugs did not modify the increase of XAB2

mobility after UV irradiation (Figure4C, green curve and Figure4—ﬁgure supplement 1D) demon-

strating that variations in XAB2 mobility after DNA damage are triggered and sustained by transcrip-

tional inhibition.

XAB2 is not released from the splicing complex during DNA repair

reactions

The increase of XAB2 mobility after UV- induced transcription inhibition could be explained by either

the release of XAB2 from a bigger complex and/or the release from an immobile (or nearly immobile)

substrate such as the chromatin or a DNA- related substrate.

In order to distinguish between these two possibilities, we ﬁrstly investigated whether, after DNA

damage induction, XAB2 dissociates from its splicing partner AQR by immunoprecipitating XAB2 and

AQR (Figure5—ﬁgure supplement 1A). Interestingly, XAB2 was immunoprecipitated more strongly

and consistently 1 hr post- irradiation, time that corresponds to the XAB2 mobility increase. At the

same time point, more AQR is also immunoprecipitated. No clear release of XAB2 from AQR was

observed at different time points.

In parallel, we also veriﬁed by PLA whether the binding of XAB2 to AQR was modiﬁed after UV- C

irradiation (Figure5—ﬁgure supplement 1B). Two hours after UV- C exposure, instead of a release

of XAB2 from AQR, we measured a more robust interaction (Figure 5—ﬁgure supplement 1B).

However, this stronger interaction could result from increased AQR concentration 1hr after UV irradi-

ation (Figure5—ﬁgure supplement 1C).

In conclusion, immunoprecipitation or PLA experiment showed that XAB2 is not released from the

splicing complex during DNA repair reactions.

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Figure 4. XAB2 is dynamic during TC- NER. (A) Fluorescent recovery after photobleaching (FRAP) analysis of XAB2-

GFP mobility in WT cells. Cells were treated or not with different doses of UV- C (2–16J/m

) and XAB2 mobility was

measured at different time points after UV- C exposure. The No UV condition was used to calculate the change in

bound fraction. (B) FRAP analysis of XAB2- GFP expressed in WT cells (MRC5- SV) and TC- NER- decient cells (CSA

Figure 4 continued on next page

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XAB2 is released from R-loops during DNA repair reactions

Interestingly, while trying to immunoprecipitate XAB2 interacting partners during DNA repair reac-

tions, we could observe that systematically and consistently, more XAB2 immunoprecipitated from

nuclear extracts 1–3 hr after UV- C irradiation in WT cells (Figure 5—ﬁgure supplement 1A and

Figure5A). At 16 hr post- irradiation, we observed that the amount of XAB2 immunoprecipitated was

comparable to the level observed in non- irradiated cells. Moreover, in CSA−/− and CSB−/− cell lines,

in which UV lesions on transcribed strands of genes are not repaired, the amount of XAB2 immunopre-

cipitated remained high all along the time course of the experiment (3 and 16hr) (Figure5A). These

results hinted that the binding between XAB2 and its substrate is not restored in TCR- deﬁcient cells.

AQR has a role in removing DNA:RNA hybrids, commonly known as R- loops structure (Sollier

et al., 2014) and recently XAB2 was found to be involved in the R- loops resolution (Goulielmaki

etal., 2021). We thus decided to investigate the possible interaction between XAB2 and R- loops.

Immunoprecipitation experiments with the S9.6 antibody show that XAB2 directly interacts with

R- loops (Figure 5B). To test the speciﬁcity of the R- loops antibody, nuclear extracts were treated

with Benzonase, an enzyme that speciﬁcally degrades DNA:RNA hybrids. Interestingly, after Benzo-

nase treatment, XAB2 is no more immunoprecipitated (Figure5B), suggesting that XAB2 substrate is

indeed DNA:RNA hybrids.

The interaction between XAB2 and R- loops was next investigated by performing IF and PLA exper-

iments with the S9.6 antibody. It has been demonstrated that the S9.6 signal is prominently cyto-

plasmic and nucleolar and derives mainly from ribosomal RNA (Smolka etal., 2021). Consequently,

to increase the speciﬁcity of the S9.6 ﬂuorescent signal, the cytoplasm was removed before ﬁxation

(Figure 5—ﬁgure supplement 2A) and during quantiﬁcation, the nucleolar signal was subtracted

from the nuclear signal (Figure5—ﬁgure supplement 2B). Using this analysis method, we observed

an increased amount of R- loops in cells silenced for XAB2 or AQR compared to control cells (Figure5C

and Figure5—ﬁgure supplement 3A).

Subsequently, we examined whether XAB2 is released from R- loops after DNA damage induc-

tion by performing a PLA assay. After UV irradiation, we measured a strong and consistent reduc-

tion of more than 40% of the interactions between R- loops and XAB2 and between R- loops and

AQR (Figure5D and Figure5—ﬁgure supplement 3B). These reduced interactions are not caused

by a reduction in either R- loops, XAB2, or AQR concentration during DNA repair (Figure5E and

Figure5—ﬁgure supplement 3B). To verify that this result is speciﬁc for R- loops and not coming

from a nonspeciﬁc interaction of XAB2 with RNAs, we performed the same assays (PLA and IF) in the

presence of RNAseH which speciﬁcally degrades R- loops structures and not single- stranded RNA

(Figure5—ﬁgure supplement 4A, B). Our results show that RNAseH reduced the quantity of R- loops

(Figure5—ﬁgure supplement 4C,F) and in doing so, it also decreased the interaction with both

XAB2 (Figure5—ﬁgure supplement 4C, D) and AQR (Figure5—ﬁgure supplement 4E, F). Next,

we veriﬁed whether the increase in XAB2 mobility observed after UV irradiation is due to a decreased

interaction with messenger RNA (mRNA) (Figure5—ﬁgure supplement 5). PLA results show that

a reduction in the interaction between XAB2 and mRNA was observed (Figure 5—ﬁgure supple-

ment 5A, C). However, this reduction paralleled the decrease of mRNA caused by UV- dependent

−/− and CSB−/−). Cells were treated or not with 8J/m

of UV- C. The No UV condition of the WT cell lines was

used to calculate the change in bound fraction. (C) FRAP analysis of XAB2- GFP mobility in WT cells after treatment

with 100µg/ml of DRB for 2hr (red line) or with 10J/m

of UV- C for 3hr (blue line) or nothing (dark curve). Inhibitor

of ATR pathway was added at 10µM in the medium 1hr before irradiation (green line). For all graphs, error bars

represent the standard error of the mean (SEM) obtained from at least 10 cells.

The online version of this article includes the following source data and gure supplement(s) for gure 4:

Source data 1. Source data for Figure4A: FRAP XAB2- GFP with different doses of UV- C.

Source data 2. Source data for Figure4B: FRAP XAB2- GFP in different cell lines.

Source data 3. Source data for Figure4C: FRAP XAB2- GFP after different treatments.

Figure supplement 1. FRAP of XAB2- GFP.

Figure supplement 1—source data 1. Source data for Figure4—ﬁgure supplement 1D: FRAP XAB2- GFP after

different treatments.

Figure 4 continued

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Figure 5. XAB2 and AQR are released from R- loops during DNA repair. (A) Immunoprecipitation (IF) of XAB2

in nuclear extract from different cell lines treated with 10J/m

of UV- C at different times. Bound proteins were

revealed by Western blotting with antibodies against XAB2. INPUT, 10% of the lysate used for IP reaction. (B) IP

of R- loops in non- crosslinked chromatin extract from WT cells treated or not with Benzonase. XAB2 bounds to

Figure 5 continued on next page

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transcription inhibition (Figure5—ﬁgure supplement 5B, C), suggesting that the results observed in

the PLA XAB2- mRNAs are due mainly to a decrease in mRNAs amount (Figure5—ﬁgure supplement

5A).

These results clearly demonstrate that XAB2 is released from R- loops during DNA repair reactions.

XAB2 is released from CSA and XPG during DNA repair

Because XAB2 has been found to participate speciﬁcally in TCR- NER repair reactions (Figure1C), we

wanted to investigate whether part of the increased XAB2 mobility observed after UV induction was

due to a release from repair complexes. We measured, by PLA, the interactions between XAB2 and

CSA, CSB, XPB, or XPG proteins, during TC- NER. Among all the proteins tested, we could observe a

clear and consistent release from the CSA protein 2hr after UV irradiation (Figure6A) and from the

XPG protein 1hr after UV irradiation (Figure6C). The corresponding IF did not show a decreased

quantity of CSA or XPG (Figure6B, D) which validated the speciﬁcity of the XAB2- CSA and XAB2- XPG

decreased interactions at those time points. On the contrary, no clear reduction of interaction was

observed between XAB2 and CSB (Figure6—ﬁgure supplement 1A, B) or between XAB2 and XPB

(Figure6—ﬁgure supplement 1C, D).

XAB2 depletion modiﬁes RNAP2 behavior

Because XAB2 was found to interact with RNAP2 (Kuraoka etal., 2008; Nakatsu etal., 2000) and

because its increased mobility after irradiation depends on the UV transcription inhibition step, we

R- loops was revealed by Western blotting. INPUT, 10% of the lysate used for IP reaction. (C) Quantication of IF

against R- loops in WT cells treated with siRNAs against indicated factors. Quantication of uorescent signal in

the nucleus against the couple XAB2_R- loops or AQR_R- loops from PLA experiments (D) or from the IF done in

parallel to PLA assays (E). Error bars represent the standard error of the mean (SEM) obtained from at least 50 cells.

P- value of Student’s test compared to No UV or siMock condition: **<0.01; ***<0.001.

The online version of this article includes the following source data and gure supplement(s) for gure 5:

Source data 1. Source data for Figure5C: quantication of IF R- loops in silenced cells.

Source data 2. Source data for Figure5D, E: quantication of PLA and IF XAB2_R- loops and AQR_R- loops.

Source data 3. Figures with the uncropped blots and relevant bands clearly labeled for Figure5A: Western blot

of IP XAB2.

Source data 4. The original les of the full raw unedited gels for Figure5A: Western blot of IP XAB2.

Source data 5. Figures with the uncropped blots and relevant bands clearly labeled for Figure5B: Western blot

of IP R- loops.

Source data 6. The original les of the full raw unedited gels for Figure5B: Western blot of IP R- loops.

Figure supplement 1. Interaction of XAB2 with the splicing complex AQR after UV damage.

Figure supplement 1—source data 1. Source data for Figure5—ﬁgure supplement 1B, C: quantication of

PLA and IF XAB2_AQR.

Figure supplement 1—source data 2. Figures with the uncropped blots and relevant bands clearly labeled for

Figure5—ﬁgure supplement 1A: Western blot of IP XAB2 in MRC5.

Figure supplement 1—source data 3. The original les of the full raw unedited gels for Figure5—ﬁgure

supplement 1A: Western blot of IP XAB2 in MRC5.

Figure supplement 2. IF R- loops and quantication method.

Figure supplement 3. Representatives images of Figure5C,D,E.

Figure supplement 4. Specicity of R- loops antibody.

Figure supplement 4—source data 1. Source data for Figure5—ﬁgure supplement 4A: quantication of PLA

and IF R- loops_RNA.

Figure supplement 4—source data 2. Source data for Figure5—ﬁgure supplement 4C, E: quantication of

PLA and IF XAB2_R- loops and AQR_R- loops after treatment with RNAseH.

Figure supplement 5. Interaction of XAB2 with RNA.

Figure supplement 5—source data 1. Source data for Figure5—ﬁgure supplement 5A,B: quantication of PLA

and IF XAB2_RNA.

Figure 5 continued

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wanted to explore whether XAB2 depletion might inﬂuence the overall RNAP2 mobility. In order

to investigate this point, we performed FRAP experiments on RNAP2- GFP expressing cells in the

presence or in the absence of XAB2 (Figure 7A; Donnio et al., 2019). Interestingly, depletion of

XAB2 signiﬁcantly increased the mobility of RNAP2 (Figure7A, dark red curve vs. dark blue curve),

demonstrating that XAB2 maintains RNAP2 bound to its substrate during transcription. Interestingly,

after UV irradiation, RNAP2 mobility did not change signiﬁcantly (p- value superior at 0.05), both in the

presence and absence of XAB2 (Figure7A, light curve vs. dark curve).

Because FRAP experiments might not reveal more subtle changes in protein–substrate interac-

tions, we decided to examine whether the absence of XAB2 might affect the contacts of RNAP2 with

Figure 6. XAB2 is released from CSA and XPG during DNA repair. Quantication of uorescent signal in the nucleus against the couple XAB2- CSA (A,

B) and XAB2- XPG (C, D) from PLA experiment (A, C) or from the IF done in parallel to PLA assay (B, D). Error bars represent the standard error of the

mean (SEM) obtained from at least 80 cells. P- value of Student’s test compared to No UV condition: *<0.05; **<0.01; ***<0.001.

The online version of this article includes the following source data and gure supplement(s) for gure 6:

Source data 1. Source data for Figure6A, B: quantication of PLA and IF XAB2- CSA.

Source data 2. Source data for Figure6C, D: quantication of PLA and IF XAB2- XPG.

Figure supplement 1. Interaction of XAB2 with CSB or XPB, repair factors of NER.

Figure supplement 1—source data 1. Source data for Figure6—ﬁgure supplement 1A, B: quantication of PLA and IF XAB2- CSB.

Figure supplement 1—source data 2. Source data for Figure6—ﬁgure supplement 1C, D: quantication of PLA and IF XAB2- XPB.

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Figure 7. RNAP2 behavior is modied without XAB2. (A) FRAP analysis of RNAP2- GFP expressing WT cells treated

or not with UV- C (10J/m

) after siRNA- mediated knockdown of the indicated factors. Error bars represent the SEM

obtained from at least 10 cells. (B, C, D) Quantication of uorescent signal in the nucleus against the couple

RNAP2_6- 4PP from PLA experiment (B) or from the IFndone in parallel to PLA assay (C, D). Error bars represent

Figure 7 continued on next page

Research article

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UV lesions using the more sensitive PLA assay. Remarkably, we measured a more robust and persistent

interaction of RNAP2 with 6- 4PP lesions after UV irradiation in XAB2- silenced cells (Figure7B and

Figure7—ﬁgure supplement 1). The corresponding IF shows a reduced increase of 6- 4PP in XAB2-

silenced cells compared to control cells (Figure7C and Figure7—ﬁgure supplement 1) while RNAP2

quantity decreases similarly during DNA repair in the presence or absence of XAB2 due to general

RNAP2 UV- dependent degradation (Figure7D and Figure7—ﬁgure supplement 1). These results

demonstrate that, without XAB2, DNA is not properly repaired and as a consequence, RNAP2 inter-

acts longer with UV lesions.

Discussion

Helix- distorting lesions continuously challenge cell survival by interfering with and blocking funda-

mental cellular functions, such as transcription and replication. In order to prevent the deleterious

effects of these events, cells have developed different mechanisms to restore an undamaged DNA

molecule and allow the restart of cellular processes. The importance of rapidly re- establishing

perturbed cellular functions is underlined by the presence of a repair mechanism directly coupled

with transcription, like the TC- NER.

Two phases can be distinguished during TC- NER events: (1) the actual repair reaction of the

damaged strand via the TC- NER subpathway and (2) the resumption of transcription after repair. The

experiment known as ‘RRS’ measures only the restart of transcription after DNA repair completion

and thus the involvement of a protein in the general TC- NER process. However, this assay does not

discriminate between the two phases of the TC- NER. As a consequence, proteins that are fully proﬁ-

cient in the repair reaction but fail in the transcription restart after DNA repair completion might have

the same defect in RRS as those solely deﬁcient in the DNA repair reaction.

Thus far, all studies have demonstrated the involvement of XAB2 in the TC- NER process using only

RRS experiments (Kuraoka etal., 2008; Nakatsu etal., 2000). In this study, we used a speciﬁc test

developed in- house (Mourgues etal., 2013) called TCR- UDS and thus demonstrated that, indeed,

XAB2 is solely needed for the repair reaction (Figure 1D). This is conﬁrmed by PLA experiments

showing an interaction between XAB2 and helix- distorting lesions, 6- 4PPs and CPDs, after irradiation

(Figure2 and Figure2—ﬁgure supplement 1). However, these experiments do not clearly discrim-

inate at which step of the repair reaction XAB2 may contribute. Two hypotheses are envisaged: (1)

XAB2 functions in damage recognition or (2) in the repair reaction per se.

XAB2 has been found as part of the pre- mRNA splicing complex composed of AQR, PRP19,

CCDC16, PPIE, and ISY1 (Kuraoka et al., 2008). However, none of these proteins take part in

the repair process, underlying the peculiar function of the splicing factor XAB2 in TC- NER repair

(Figure3—ﬁgure supplements 1–3).

We also demonstrated that, unlike all the other NER proteins studied so far, XAB2 protein is released

from the damaged region induced by UV- C exposure (Figure3). Concomitantly, we also observed an

increase in XAB2 cellular mobile fraction after UV irradiation (Figure 4A). This release from DNA-

damaged area and increased mobility is surprising and atypical for a repair protein. However, this

behavior has also been observed for late- stage spliceosomes (Tresini et al., 2015) and could be

explained by the importance of the cell rapidly providing access to the repair machinery.

Surprisingly, the increased XAB2 mobility occurs in the absence of CSA and CSB proteins, with

inhibitors of DDR after UV irradiation but also after transcription inhibition (Figure4 and Figure4—

ﬁgure supplement 1). These results strongly suggest that XAB2 remobilization is independent of

the repair process but it is more a result of the transcription inhibition induced by the DNA damage.

the standard error of the mean (SEM) obtained from at least 80 cells. P- value of Student’s test compared to No UV

condition: *<0.05; ***<0.001.

The online version of this article includes the following source data and gure supplement(s) for gure 7:

Source data 1. Source data for Figure7A: FRAP RNAP2- GFP.

Source data 2. Source data for Figure7B–D: quantication of PLA and IF RNAP2_6- 4PP.

Figure supplement 1. Representatives images of Figure7B–D.

Figure 7 continued

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Moreover, the recovery of intrinsic XAB2 mobility is CSA and CSB dependent probably because in

TC- NER defective cells, repair of transcribed genes is deﬁcient and transcription is not recovered. As a

consequence, a proper repair and re- establishment of the transcription process are needed to restore

XAB2 mobility to normal values.

Previously, we reported that a complete TC- NER mechanism is required to repair the UV lesions

present on active rDNA, genes transcribed by RNAP1 (Daniel etal., 2018). Notably, both Cockayne

syndrome proteins (CSA and CSB) are implicated in this speciﬁc repair reaction, as well as the UV- stim-

ulated scaffold protein A (UVSSA), a protein required for the stabilization of CSB speciﬁcally after

UV irradiation (Higa etal., 2016). By measuring the level of ribosomal RNA, we demonstrate that

RNAP1 transcription restarts after irradiation in the absence of XAB2 (Figure2—ﬁgure supplement

2), meaning that UV lesions present on active rDNA are repaired. As a consequence, XAB2 is involved

only in TC- NER of RNAP2- transcribed genes and probably not in the repair of RNAP1- transcribed

genes, conﬁrming a likely speciﬁc interaction with RNAP2 and reinforcing the idea that RNAP1 and

RNAP2 repair processes are distinct although they share common proteins.

FRAP experiments in CSA and CSB mutant cells show a more immobile XAB2 fraction than in WT

cells (Figure4B) without any damage induction. Tanaka’s group found that XAB2 interacts in vitro

with CSA and CSB protein in the absence of DNA damage (Nakatsu etal., 2000). In addition, several

studies demonstrated the involvement of CSB in transcription regulation (Boetefuer etal., 2018). As

both CSB and XAB2 are necessary during the transcription process, it is therefore possible that the

absence of CSB will modify XAB2 mobility. However, it is not excluded that in CSA and CSB mutant

cells, a low level of unrepaired oxidative damage (de Waard etal., 2004) might interfere with the

proper XAB2 mobility, eventually modifying the amount of R- loops within these cells. Nevertheless,

it is difﬁcult to precisely estimate the exact number of R- loops between different cell types, and this

hypothesis is difﬁcult to clearly assess.

The UV- induced remobilization of XAB2 is not explained by its release from the splicing complex,

as demonstrated by co- immunoprecipitation and PLA experiments, but it is more the result of the

release from chromatin- speciﬁc structures, in this particular case the R- loops (Figure5). An R- loop is

a three- stranded nucleic acid structure composed of a DNA:RNA hybrid and the associated nontem-

plate single- stranded DNA. This structure arises naturally in organisms from bacteria to humans, and

has a multitude of functions in the cell (Belotserkovskii etal., 2018). Our results show that R- loops are

a substrate for XAB2, and after DNA damage induction, the interaction between XAB2 and R- loops is

strongly reduced. This might explain the increased XAB2 mobility during the TC- NER reaction.

It was previously demonstrated that, in AQR- depleted cells, R- loops formation is induced. These

R- loops are actively processed into DNA double- strand breaks by XPF and XPG, the NER endonu-

cleases (Sollier etal., 2014). Without any damage, we also observed an increased level of cellular

R- loops in both AQR- and XAB2- silenced cells (Figure5C), suggesting an involvement of these two

proteins in R- loops resolution, as recently also demonstrated by Goulielmaki etal., 2021.

Transcription process and R- loops formation are ﬁnely interconnected. Indeed, R- loops formation

can cause transcription blockage. Transcription blockage due to DNA damage appears to result in

R- loops formation (Mullenders, 2015; Steurer and Marteijn, 2017). Moreover, transcription activity

declines after UV irradiation and mRNAs levels are drastically reduced (Figure5—ﬁgure supplement

5). PLA assays show that, after UV irradiation, XAB2 and total RNAs interaction is reduced. However,

because the total amount of RNAs is diminished after transcription block, we assume that XAB2_RNAs

interaction is lessened because of the intrinsic reduced amount of RNAs. Differently from total RNAs,

in our study, we observe that R- loops do not decrease in number after UV damage and transcrip-

tion inhibition (Figure5E). Therefore, the interaction XAB2_R- loops and AQR_R- loops is speciﬁcally

hindered after UV damage (Figure5D). However, a careful interpretation of these results should be

made because a recent paper demonstrates that the S9.6 antibody, speciﬁc for DNA:RNA hybrids,

can also recognize other nucleic acid structures in immunoﬂuorescence assay (Smolka etal., 2021). To

be able to conﬁrm our results, many additional controls were performed: (1) removal of the cytoplasm

before ﬁxation (Figure5—ﬁgure supplement 2A); (2) subtraction of the nucleolar signal from the

nuclear signal (Figure5—ﬁgure supplement 2B); (3) treatment with RNAseH which degrade specif-

ically R- loops (Figure5—ﬁgure supplement 4); and (4) ﬁnally interaction of XAB2 with nascent RNA

(EU staining) is different from interaction with R- loops (Figure5—ﬁgure supplement 5). Although all

controls point to a seeming interaction between XAB2 and R- loops or between AQR and R- loops, we

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cannot exclude that XAB2 and AQR do not release from DNA:RNA hybrids but from other nucleic acid

structures, for example, double- stranded DNA.

Because XAB2 interacts in vitro with CSA and CSB protein (Nakatsu et al., 2000) and recent

studies have shown an interaction with XPG (Goulielmaki etal., 2021), we decided to verify whether

these interactions were modiﬁed during the DNA repair process and the concomitant transcription

inhibition. Our results show a clear and consistent reduction of interaction between XAB2 and CSA

(Figure 6A, B) and between XAB2 and XPG (Figure 6C, D) but not between XAB2 and CSB or

between XAB2 and XPB (Figure6—ﬁgure supplement 1). These results suggest that XAB2 inter-

venes in the early steps of TC- NER or at least in the steps that imply the activity of CSB and TFIIH and

most probably the early RNAP2 blocking- recognition step. However, the reduction of interactions

between XAB2 and CSA or XPG is less striking than the one with R- loops. As suggested in Gouliel-

maki etal., 2021, another working hypothesis would be that XAB2 is released from CSA and XPG

proteins associated with R- loops processing.

The physical interaction between XAB2 and RNAP2 has already been established (Kuraoka etal.,

2008; Nakatsu et al., 2000), but the exact relation between XAB2 and RNAP2 has not yet been

disclosed. Without DNA damage, we observed a difference in nascent RNA synthesis in XAB2- silenced

cells compared to control cells (Figure1—ﬁgure supplement 2B). Since XAB2 functions in both tran-

scription and splicing, it is not surprising that the quantity of nascent RNA is altered in the absence of

XAB2 (Goulielmaki etal., 2021; Kuraoka etal., 2008). Moreover, we have clearly demonstrated by

FRAP experiments that RNAP2 mobility is severely affected in the absence of XAB2. Namely, in XAB2-

depleted cells, RNAP2 is released from its substrate and its mobility is strongly increased (Figure7A).

RNAP2 immobile fraction after UV irradiation does not change signiﬁcantly in the presence or absence

of XAB2. However, we could observe that interactions of RNAP2 with the UV lesions (6- 4PPs or CPDs)

Figure 8. Model of XAB2 dynamics during DNA damage- dependent transcription inhibition. Considering our results, a hypothetical model of XAB2

roles and dynamics can be sketched. XAB2 is involved in R- loops removal and pre- mRNA splicing, both processes linked to transcription. After DNA

damage induction, transcription is blocked and XAB2 (together with some of the proteins involved in the splicing) is massively released from R- loops

allowing a subset of XAB2 molecules to interact with UV- stalled RNA polymerase 2 (RNAP2) and participate in the TC- NER process. In the absence of

XAB2, TC- NER is defective and as a consequence, RNAP2 remains longer in the proximity of DNA lesions.

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are stronger after DNA damage and last longer in siXAB2- treated cells compared to siMock- treated

cells (Figure7B–D). These results suggest that, during transcription, XAB2 helps RNAP2 anchoring to

its substrate, whereas during the TC- NER process, it has an effect on stalling of RNAP2 on UV lesions,

advocating for a potential role in the DNA damage recognition step.

In conclusion, we describe here an increased mobility of the protein XAB2 during the DNA damage-

dependent transcription inhibition. This increased mobility might partly be explained by the release of

XAB2 from its substrate R- loops and its partner CSA and XPG. Importantly, we demonstrate that XAB2

plays an anchoring role for RNAP2 to its substrate during transcription and helps RNAP2 to detach

from UV lesions after DNA damage. As aconsequence, the absence of XAB2 hinders the overall tran-

scription activity of the cells and severely affects the TC- NER capacity (Figure8).

Materials and methods

Cell culture and treatments

The cells used in this study come from Erasmus MC in Rotterdam and were: (1) wild- type SV40-

immortalized human ﬁbroblasts (MRC5 [RRID:CVCL_D690]); (2) XPC- deﬁcient SV40- immortalized human

ﬁbroblast (XP4PA- SV, GG- NER deﬁcient [RRID:CVCL_6E33]); (3) CSA- deﬁcient SV40- immortalized human

ﬁbroblast (CS3BE, TC- NER deﬁcient [RRID:CVCL_F631]); (4) CSB- deﬁcient SV40- immortalized human

ﬁbroblast (CS1AN, TC- NER deﬁcient [RRID:CVCL_L472]); (5) MRC5- SV stably expressing XAB2- GFP; (6)

CS3BE- SV stably expressing XAB2- GFP; (7) CS1AN- SV stably expressing XAB2- GFP; and (8) MRC5- SV

stably expressing RNAP2- GFP. All cell lines are regularly tested negative for mycoplasma contamination.

Immortalized human ﬁbroblasts were cultured in Dulbecco's Modiﬁed Eagle Medium(DMEM from

Sigma) supplemented with 1% of penicillin and streptomycin (Gibco) and 10% fetal bovine serum

(Corning) and incubated at 37°C with 5% CO

DNA damage was inﬂicted by UV- C light (254nm, 6- W lamp). Cells were globally irradiated with a

UV- C dose of 2, 4, 8, 10, or 16 J/m

or locally irradiated with a UV- C dose of 60 or 100 J/m

through

a ﬁlter with holes of 5µm of diameter (Millipore). After irradiation, cells were incubated at 37°C with

5% CO

for different periods.

Inhibitor of ATR pathway (VE821) and ATM pathway (KU55933) was added at 10µM in the medium

1hr before irradiation.

Construction and expression of RNAP2-GFP and XAB2-GFP fusion

protein

Full- length RNAP2 c- DNA was cloned in- frame into the pEGFP- C1 vector (Clontech), and full- length

XAB2 cDNA was cloned in- frame into the pEGFP- N1 vector. Constructs were sequenced prior to

transfection.

XAB2- GFP and RNAP2- GFP stably expressing cell lines were produced by transfecting XAB2- GFP

or RNAP2- GFP in MRC5, CSA, or CSB cells using FuGENE 6 Transfection Reagent (Promega) according

to the manufacturer’ protocol. The selection was performed with G418 at 2mg/ml.

Transfection of small interfering RNAs

The small interfering RNA (siRNAs) used in this study are: siMock, Horizon, D- 001206- 14 (10 nM);

siXAB2, Horizon, L- 004914- 01 (20nM); siXPF, Horizon, M- 019946- 00 (10nM); siAQR, CCAG ACCA

CUUC CCAU UCU (10nM); siPRP19, GGUG UACA UGGA CAUC AAG (10nM); siCCDC16, GCGA UCUA

GUUU CAUU AAA (5nM); siPPIE, GGC UAUG AGGC AAGU CAAC (5nM); siISY1, GGAA AUCG AGGU

UACA AGU (5nM) and siCSB, Horizon, L- 004888- 00 (10nM). The ﬁnal concentration used for each

siRNA is indicated in parentheses. All siRNA from Horizon are a pool of four different siRNA.

Cells were seeded in 6- well plates and allowed to attach for at least 24hr. Coverslips were added

inside the well if needed for the experiment. Cells were transfected two times with an interval of 24

hr with siRNA using Lipofectamine RNAiMAX reagent (Invitrogen; 13778150) or GenJet (Tebu- Bio),

according to the manufacturer’s protocol. Experiments were performed between 24 and 72hr after

the second transfection. Protein knockdown was conﬁrmed for each experiment by Western blot.

RRS assay

MRC5 cells were grown on 18mm coverslips. siRNA transfections were performed 24 and 48hr before

the RRS assay. RNA detection was done using a Click- iT RNA Alexa Fluor Imaging kit (Invitrogen),

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according to the manufacturer’s instructions. Brieﬂy, cells were UV- C irradiated (10J/m²) and incu-

bated for 0, 3, 16, and 24hr at 37°C. Then, cells were incubated for 2hr with 100µM of 5- ethynyl

uridine (EU). After ﬁxation with 4% paraformaldehyde (PFA) for 15min at 37°C and permeabilization

with phosphate- buffered saline (PBS) and 0.5% Triton X- 100 for 20 min, cells were incubated for

30min with the Click- iT reaction cocktail containing Alexa Fluor Azide 594. After washing, the cover-

slips were mounted with Vectashield (Vector). Using ImageJ, the average ﬂuorescence intensity per

nucleus was estimated after background subtraction and normalized to not treated cells.

For RRS with siRNA against splicing protein, cells were incubated for 1 hr with 2.5 mM of

5- bromouracile (BrU). Next, the protocol is the same as immunoﬂuorescence. The primary antibody

used is mouse anti- BrU diluted in PBS+ (PBS containing 0.15 glycine and 0.5% bovine serum albu-

min[BSA]) at 1/750 (11170376001 [sigma]).

RNA ﬂuorescence in situ hybridization

Cells were grown on 18mm coverslips, washed with warm PBS, and ﬁxed with 4% PFA for 15min at

37°C. After two washes with PBS, cells were permeabilized with PBS + 0.4% Triton X- 100 for 7min

at 4°C. Cells were washed rapidly with PBS before incubation (at least 30 min) with prehybridiza-

tion buffer: 15% formamide in 2× SSPE (Sodium Chloride- Sodium Phosphate- EDTA) (0.3 M NaCl,

15.7mM NaH

·H

O, and 2.5mM EDAT [Ethylenediaminetetraacetic acid] et pH8.0). 35ng of the

probe was diluted in 70µl of hybridization mix (2× SSPE, 15% formamide, 10% dextran sulfate and

0.5mg/ml tRNA). Hybridization of the probe was conducted overnight at 37°C in a humidiﬁed envi-

ronment. Subsequently, cells were washed twice for 20min with prehybridization buffer and once for

20min with 1× SSPE. After extensive washing with PBS, the coverslips were mounted with Vectashield

containing DAPI (Vector). The probe sequence (5′ to 3′) is Cy5- AGAC GAGA ACGC CTGA CACG CACG

GCAC .

UDS or TCR-UDS

MRC5- SV (WT) or XP4PA- SV (GG- NER- deﬁcient) cells were grown on 18mm coverslips. siRNA trans-

fections were performed 24 and 48hr before UDS assays. After local irradiation at 100 J/m

with UV- C

through a 5µm pore polycarbonate membrane ﬁlter, cells were incubated for 3 or 8hr (UDS and

TCR- UDS, respectively) with 20µM of EdU (5- ethynyl- 2′-deoxyuridine), ﬁxed with 4% PFA for 15min

at 37° C and permeabilized with PBS and 0.5% Triton X- 100 for 20min. Then, cells were blocked with

PBS+ (PBS, 0.15% glycine and 0.5% BSA) for 30min and subsequently incubated for 1hr at room

temperature (RT) with mouse monoclonal anti-γH2AX antibody (Ser139 [Upstate, clone JBW301])

1:500 diluted in PBS+. After extensive washes with PBS containing 0.5% Triton X- 100, cells were incu-

bated for 45min at RT with secondary antibodies conjugated with Alexa Fluor 594 ﬂuorescent dyes

(Molecular Probes, 1:400 dilution in PBS+). Next, cells were washed several times and then incubated

for 30min with the Click- iT reaction cocktail containing Alexa Fluor Azide 488. After washing, the

coverslips were mounted with Vectashield containing DAPI (Vector). Images were analyzed as follows

using ImageJ and a circle of constant size for all images: (1) the background signal was estimated in

the nucleus (avoiding the damage, nucleoli, and other nonspeciﬁc signals) and subtracted, (2) the

locally damaged area was deﬁned by using the γH2AX staining, and (3) the average ﬂuorescence

correlated to the EdU incorporation was then measured and thus an estimation of DNA synthesis after

the repair was obtained.

Immunoﬂuorescence

Cells were plated on 12 or 18mm coverslips to reach 70% conﬂuence on the day of the staining. After

two washes with PBS, cells were ﬁxed with 2% PFA for 15min at 37°C. Cells were permeabilized by

three short washes followed by two washes of 10min with PBS + 0.1% Triton X- 100. Blocking of the

nonspeciﬁc signal was performed with PBS+ (PBS, 0.5% BSA, 0.15% glycine) for at least 30min. Then,

coverslips were incubated with primary antibody diluted in PBS+ for 2hr at RT or overnight at 4°C

in a moist chamber. After several washes with PBS + 0.1% Triton X- 100 (three short washes and two

of 10min) and a short wash with PBS+, cells were incubated for 1hr at RT in a moist chamber with a

secondary antibody coupled to a ﬂuorochrome (Goat anti- mouse Alexa Fluor 488 [A11001, Invitrogen]

or 594 [A11005] and Goat anti- rabbit Alexa Fluor 488 [A11008] or 594 [A11012], 1/400 dilution in

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PBS+). After the same washing procedure with PBS instead of PBS + 0.1% Triton X- 100, coverslips

were ﬁnally mounted using Vectashield with DAPI (Vector Laboratories).

Treatment with RNAseH (NEB, M0297L) was performed after blocking with PBS+. RNAseH was

diluted in PBS+ to put 6U per coverslip and incubated for 1hr at 37°C. After washing with PBS+ (one

quick and one of 10min), a primary antibody was added.

For local damage immunoﬂuorescence, the variation of ﬂuorescence in the locally irradiated zone

has been calculated, as for the UDS experiment.

For Immunoﬂuorescence with UV lesions antibodies (6- 4PP and CPD), a step of DNA denaturation

with 0.07M NaOH freshly diluted in PBS for 5min at RT was added after permeabilization. After

several washes with PBS + 0.1% Triton X- 100 (three short washes and two of 10min), cells were incu-

bated with primary antibody.

For RNA detection, the protocol was adapted from Petruk etal., 2016. Brieﬂy, cells were incu-

bated for 2hr with 100µM of EU. After ﬁxation, permeabilization, and blocking, a Biotin tag was

added thanks to a click- it reaction and then recognized by an antibody.

Proximity Ligation Assay

PLA experiments were done using Duolink II secondary antibodies and detection kits (Sigma- Aldrich,

#DUO92002, #DUO92004, and #DUO92008) according to the manufacturer’s instructions. The cells

were ﬁxed and permeabilized with the same procedure as immunoﬂuorescence. After blocking 1hr

at 37°C with the Blocking Solution from the kit, the primary antibodies diluted in Antibody Diluent

was incubated at 4°C overnight. After one quick and three washes of 5 min with PLA buffer A,

Duolink secondary antibodies were added and incubated for 1hr at 37°C. After the same washing

procedure with PLA buffer A, if secondary antibodies were in close proximity (<40nm), they were

ligated together to make a closed circle thanks to the incubation of 30min at 37°C with the Duolink

ligation solution. Then, after the same washing procedure, the DNA is ampliﬁed and detected by

ﬂuorescence 594 thanks to the incubation of 100min at 37°C with the Duolink ampliﬁcation solu-

tion. After washing with PLA buffer B, coverslips were mounted using Vectashield with DAPI (Vector

Laboratories).

Cytostripping

To remove the background generated by some antibodies or EdU incorporation, the cytoplasm of

the cells was removed before ﬁxation. After two washes with cold PBS, cells were incubated on ice

5min with cold cytoskeleton buffer (10mM PIPES [piperazin- N,N'-bis(2- ethanesulfonic acide)] pH 6.8;

100mM NaCl; 300mM sucrose; 3mM MgCl

; 1mM EGTA [egtazic acid]; 0.5% Triton X- 100) followed

by 5 min with cold cytostripping buffer (10mM Tris–HCl pH 7.4; 10mM NaCl; 3mM MgCl

; 1% Tween

40; 0.5% sodium deoxycholate). After three gentle washes with cold PBS, cells were ﬁxed.

Images acquisition and analysis

For RRS, images of the cells were obtained using an Andor spinning disk: Olympus IX 83 inverted

microscope, equipped with a Yokaga CSU- X1 Spinning disk Unit and BOREALIS technology for homo-

geneous illumination. The acquisition software is IQ3.

For RNAFish, UDS, TCR- UDS, and IF of splicing complex after local damage, images of the cells

were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following

objective: Plan- Apochromat ×63/1.4 oil DIC (Differential Interference Contrast) M27 or ×40/1.3 oil

DIC. The acquisition software is ZEN.

PLA and IF associated with PLA have been performed on a Zeiss Z1 imager right using a ×40/0.75

dry objective. The acquisition software is Metavue.

Images of the cells for each experiment were obtained with the same microscopy system and

constant acquisition parameters. All images were analyzed with ImageJ software. All experiments

have been performed at least two times and are biological replicates.

Error bars represent the standard error of the mean of the biological replicates. Excel was used for

statistical analysis and plotting of all the numerical data. Statistics were performed using a Student’s

test to compare two different conditions (siMock vs. siRNA X or No UV vs. after irradiation) with the

following parameters: two- tailed distribution and two- sample unequal variance (heteroscedastic).

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Primary antibodies used for IF and PLA

Primary antibodies used for immunoﬂuorescence and PLA experiments were anti- 6- 4PP (mouse,

NM- DND- 002 [Cosmobio] 1/500 dilution), anti- CPD (mouse, NM- DND- 001 [cosmobio], 1/200 dilu-

tion), anti- XAB2 (mouse, sc- 271037 [Santa Cruz Biotechnology], 1/1000 dilution and rabbit, A303- 638A

[Béthyl], 1/500 dilution), anti- AQR (IPB160 rabbit, A302- 547A [Béthyl], 1/500 dilution), anti- CCDC16

(rabbit, HPA027211 [atlas antibodies], 1/250 dilution), anti- PRP19 (rabbit, ab27699 [abcam], 1/500

dilution), anti- DNA:RNA hybrid clone S9.6 (mouse, MABE1095 [Merck Millipore], 1/100 dilution and

rabbit, Ab01137- 23.0 [Absolute antibody], 1/100 dilution), anti- Biotin (rabbit, ab1227 [abcam] 1/1000

dilution), anti- CSA (rabbit, GTX100145 [genetex], 1/400 dilution), anti- and anti- CSB (mouse, sc398022

[santa- cruz], 1/200 dilution), anti- XPB (rabbit, sc293 [santa- cruz], 1/500 dilution), and anti- XPG (rabbit,

sc84663 [santa- cruz], 1/1000 dilution).

Fluorescence recovery after photobleaching

FRAP experiments were performed on a Zeiss LSM 780 NLO confocal laser scanning microscope

(Zeiss), using a ×40/1.3 oil objective under a controlled environment (37°C, 5% CO

). A narrow region

of interest (ROI) centered across the nucleus of a living cell was monitored every 20 ms (1% laser inten-

sity of the 488- nm line of a 25- mW Argon laser) until the ﬂuorescence signal reached a steady state

level (after ≈2s). The same region was then photobleached for 20 ms at 100% laser intensity. Recovery

of ﬂuorescence in the bleached ROI was then monitored (1% laser intensity) every 20 ms for about

20s. Analysis of raw data was performed with the ImageJ software. All FRAP data were normalized to

the average prebleached ﬂuorescence after background removal.

XAB2- GFP SPOT FRAP data were analyzed as follows (Figure4—ﬁgure supplement 1). The No

UV condition’s average ﬂuorescence (over all cells) was subtracted from the average ﬂuorescence of

the UV- treated conditions. The obtained difference between the two FRAP curves was then summed

point by point, starting from the bleach up to the following 100 measurements, that is, the area

between the curve of interest and the untreated condition curve.

Protein extraction

For veriﬁcation of siRNA efﬁciency, cells were cultured in a 6- well plate. The coverslip needed for

the experiment was displaced before ﬁxation, and cells that remained in the dish were collected.

The extraction of total proteins has been performed using the PIERCE RIPA buffer (Thermo, #89900)

complemented with PIC (Protease Inhibitor Cocktail from ROCHE).

For immunoprecipitation, cells cultured in 10 cm dishes were harvested by scraping, and the

pellet was washed once with PBS supplemented with the PIC. The extraction of nuclear proteins has

been performed using the CelLytic NuCLEAR Extraction kit (Sigma- Aldrich) complemented with PIC.

Protein concentration was determined using the Bradford method. The samples were diluted with

Laemmli buffer (10% glycerol, 5% β-mercaptoethanol, 3% sodium dodecyl sulfate, 100mM Tris–HCl

[pH 6.8], bromophenol blue) and heated at 95°C before loading on a SDS- PAGE (sodium dodecyl

sulfate–polyacrylamide gel electrophoresis).

Coimmunoprecipitation

For coimmunoprecipitation, 10µl of protein G magnetic beads (Bio- adembead, Ademtech) were used

per IP. 1µg of anti- XAB2 antibody (rabbit, A303- 638A, Bethyl) were bound to the beads in PBS with

3% BSA 3% for 2hr at 4°C with rotation. 100µg of nuclear extracts were then incubated with beads–

antibodies complex for 2hr at 4°C with rotation. After two washes at 100mM salt, two at 150mM,

and one wash at 100mM, beads were boiled in 2× Laemmli buffer and eluted samples loaded on a

SDS–PAGE.

RNA/DNA hybrid IP

Non- crosslinked MRC5 cells were lysed in 85mM KCl, 5 mM PIPES (pH 8.0), and 0.5% NP- 40 for

10min on ice. Pelleted nuclei were resuspended in RSB buffer (10 mM Tris–HCl pH 7.5, 200 mM

NaCl, 2.5mM MgCl

) with 0.2% sodium deoxycholate, 0.1% SDS, 0.05% sodium lauroyl sarcosinate,

and 0.5% Triton X- 100, and extracts were sonicated for 10min (Diagenode Bioruptor, 60 cycles high

power, 10s ON and 20s OFF). Extracts were then diluted 1:4 in RSB with 0.5% Triton X- 100 (RSB- T)

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and subjected to IP with the S9.6 antibody overnight at 4°C. RNaseA was added during IP at 0.1ng

RNaseA per microgram genomic DNA. Then protein G dynabeads (Invitrogen) washed with RSB- T

were added and incubated for 3hr. Beads were washed 4× with RSB- T and 2× with RSB; then eluted in

2× Laemmli buffer for 10min at 95°C before loading on SDS–PAGE. When indicated, nuclear extracts

were treated with 0.25U/µl Benzonase (Sigma 70664) for 30min at 37°C before IP.

Western blot

Proteins were separated on a SDS–PAGE composed of bisacrylamide (37:5:1), blotted onto a PVDF

(polyvinylidene diﬂuoride) membrane (0.45μm Millipore). The membrane was blocked in PBS- T (PBS

and 0.1% Tween 20) with 5% milk and then incubated for 2hr at RT or overnight at 4°C with the

following primary antibodies diluted in milk PBS- T: Rabbit anti- XAB2, A303- 638A (Bethyl) 1/1000;

Mouse anti- XPF, MS- 1351- P1 (NeoMarkers) 1/500; Mouse anti-α-Tubulin, T6074 (Sigma- Aldrich)

1/50,000; Rabbit anti- AQR (A302- 547A [Bethyl] 1/2000); Rabbit anti- CCDC16 (A301- 419A [Bethyl]

1/2000), Rabbit anti- PPIE (ab154865 [abcam] 1/1000); Rabbit anti- PRP19 (ab27692 [abcam] 1/1000);

Rabbit anti- ISY1 (ab121523 [abcam] 1/500); Mouse anti- UBF (sc13125 [santa- cruz] 1/500); and Goat

anti- CSB (sc10459 [santa- cruz] 1/100).

Subsequently, the membrane was washed repeatedly with PBS- T and incubated 1hr at RT with

the following secondary antibody diluted 1/5000 in milk PBS- T: Goat anti- rabbit IgG HRP conjugate

(170- 6515; BioRad), Rabbit anti- goat IgG HRP conjugate (172- 1034, BioRad) or Goat anti- mouse IgG

HRP conjugate (170- 6516; BioRad). After the same washing procedure, protein bands were visualized

via chemiluminescence (ECL Enhanced Chemiluminescence; Pierce ECL Western Blotting Substrate)

using the ChemiDoc MP system (BioRad).

Acknowledgement

Additional information

Funding

Funder Grant reference number Author

Agence Nationale de la

Recherche

ANR-14-CE10-0009 Giuseppina Giglia-Mari

Institut National Du Cancer PLBIO17-043 Giuseppina Giglia-Mari

Institut National Du Cancer PLBIO19-126 Giuseppina Giglia-Mari

Ligue Contre le Cancer 218398 Giuseppina Giglia-Mari

Electricité de France 218398 Giuseppina Giglia-Mari

The funders had no role in study design, data collection, and interpretation, or the

decision to submit the work for publication.

Author contributions

Lise- Marie Donnio, Data curation, Formal analysis, Validation, Investigation, Visualization, Method-

ology, Writing – original draft, Writing – review and editing; Elena Cerutti, Conceptualization, Formal

analysis, Validation, Investigation, Methodology, Writing – original draft; Charlene Magnani, Damien

Neuillet, Investigation, Methodology; Pierre- Olivier Mari, Data curation, Software, Supervision,

Writing – review and editing; Giuseppina Giglia- Mari, Conceptualization, Supervision, Funding acqui-

sition, Validation, Writing – original draft, Project administration, Writing – review and editing

Author ORCIDs

Lise- Marie Donnio

http://orcid.org/0000-0002-2414-6034

Elena Cerutti

http://orcid.org/0000-0002-4644-4817

Giuseppina Giglia- Mari

http://orcid.org/0000-0003-2001-1965

Research article

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Donnio, Cerutti etal. eLife 2022;0:e77094. DOI: https://doi.org/10.7554/eLife.77094 23 of 24

Decision letter and Author response

Decision letter https://doi.org/10.7554/eLife.77094.sa1

Author response https://doi.org/10.7554/eLife.77094.sa2

Additional ﬁles

Supplementary ﬁles

• MDAR checklist

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting ﬁle.

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