# Repair of DNA Double-Strand Breaks in RNAPI-and RNAPII-Transcribed loci

E. Lesage, T. Clouaire, G. Legube

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## To cite this version:

E. Lesage, T. Clouaire, G. Legube. Repair of DNA Double-Strand Breaks in RNAPI-and RNAPII-Transcribed loci. DNA Repair, 2021, 104, pp.103139. 10.1016/j.dnarep.2021.103139. hal-04832588

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Distributed under a Creative Commons Attribution 4.0 International License
Repair of DNA Double-Strand Breaks in RNAPI- and RNAPII-Transcribed loci

Lesage E¹, Clouaire T¹ and Legube G¹*

¹Molecular, Cellular and Developmental biology unit (MCD), Centre de Biologie Intégrative (CBI), UPS, CNRS, Toulouse, France

*Corresponding author gaelle.legube@univ-tlse3.fr
Abstract

DNA double-strand Breaks (DSBs) are toxic lesions triggered not only by environmental
sources, but also by a large number of physiological processes. Of importance,
endogenous DSBs frequently occur in genomic loci that are transcriptionally active.
Recent work suggests that DSBs occurring in transcribed loci are handled by specific
pathway(s) that entail local transcriptional repression, chromatin signaling, the
involvement of RNA species and DSB mobility. In this *Graphical Review* we provide an
updated view of the “Transcription-Coupled DSB repair” (TC-DSBR) pathway(s) that are
mounted at DSBs occurring in loci transcribed by RNA Polymerase I (RNAPI) or RNA
Polymerase II (RNAPII), highlighting differences and common features, as well as yet
unanswered questions.
DNA double-strand Breaks (DSBs) are toxic lesions that occur for instance following exposure to radiation or chemotherapies. They also arise on the genome owing to endogenous processes, such as replication stress and topoisomerase reactions. Of importance, the occurrence of these endogenous DSBs display a clear bias for accessible chromatin and transcribed loci, which are more prone to form DNA secondary structures and accumulate torsional stress[1]. DSBs are mainly repaired by two rather well characterized pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), yet a plethora of less well defined alternative repair mechanisms has been described[2].

Execution of these DSB repair pathways is known to be regulated by the cell cycle but was recently found to be also strongly influenced by the genomic position of the DSB. Indeed, the exponential availability of new tools to induce DSBs at determined locations on the genome (eg: CRISPR/Cas9, TALEN, AsISI, I-Ppol etc..) has provided means to study repair mechanisms that take place in heterochromatin, on telomeric sequences, genes and ribosomal DNA (rDNA)[3]. Importantly, recent work pinpoints the existence of specialized repair pathways handling DSBs occurring in transcribing loci (Transcription-Coupled DSBs, or TC-DSBs), collectively referred to as Transcription-Coupled DSB repair (TC-DSBR)[4] and that we aim to synthesize here. We will describe the main features of TC-DSBR as well as specific differences when this pathway takes place at loci transcribed by RNA polymerase I (RNAPI i.e. rDNA) and RNA polymerase II (RNAPII).

# 1. Local transcriptional repression

One of the earliest response to a DSB in a transcribing locus is the rapid and local repression of the gene(s) nearby (Fig. 1).

*RNAPII-transcribed loci*
DSBs that arise within or at the immediate vicinity of RNAPII-transcribed genes elicit a local
transcription shut-down that depends on the protein kinases ATM, DNAPK and DYRK1B, and on
Poly-ADP Ribose Polymerase 1 (PARP1)[5,6]. These enzymatic activities directly and indirectly
trigger modifications of both the transcription machinery and chromatin structure, which
altogether establish a repressive environment for transcription initiation and elongation. The
chromatin assembled at promoters undergoes a transition from “active” to “inactive” histone
marks. Accumulation of the bromodomain-containing protein ZMYND8 at DSBs recruits the
histone demethylase KDM5A, ensuring demethylation of H3K4me3, and the NURD complex,
allowing for histone deacetylation[7,8]. Moreover, multiple reports also underlined the prime
importance of the polycomb pathway in this transcriptional shutdown[1,5]: ATM triggers the
phosphorylation of the AF9 subunit of the super-elongation complex. pAF9 further directly
interacts with PRC1 subunit and Ring1b/Bmi1, that induce H2A ubiquitination on lysine K119,
hence triggering a shift from elongation to repression. Moreover, the PcG protein EZH2 is also
recruited at the vicinity of DSB, catalyzing H3K27 tri-methylation. Finally, the kinase DYRK1B
triggers the recruitment of the histone H3 methyltransferase G9a[6]. Altogether these histones
modifications collectively ensure that a repressive context is established locally around DSBs.
Beyond histone marks, the cohesin complex, which is a main player in chromatin architecture
thanks to its loop extrusion activity, is also required to ensure local transcription shut-down post-
DSB although the precise mechanism remains unclear[9].

Besides, the RNAPII transcription cycle itself is altered following TC-DSB: RNAPII is parylated,
and PARP1 is required to recruit the pausing factor NELF-E at TC-DSB[10], which likely inhibits
the release of promoter-paused RNAPII into gene bodies. This may account for the reported
reduction of the elongating form of RNAPII (phosphorylated on CTD serine 2) on genes
bodies[11,12]. On the other hand, WWP2 triggers K48-linked polyubiquitylation of RNAPII,
further leading to a DNA-PK- and proteasome-dependent premature eviction of RNAPII from the
gene body[13]. The nuclear exosome is also recruited presumably to terminate transcription and degrade aborted pre-mRNAs[14,15].

### RNAPI-transcribed loci

Induction of DSB in the ribosomal DNA also leads to the rapid and transient extinction of the surrounding RNAPI-dependent RNA synthesis. This process also strongly relies on the activity of ATM and DYRK1B, but is surprisingly independent of PARP1 and DNA-PK[16,17]. As for RNAPII genes, the cohesin complex is necessary to ensure transcriptional repression of damaged rDNA[18]. Cohesin recruits the HUSH complex, comprising of periphilin, MPP8 and in the case of rDNA, Suv39h1/2, which triggers deposition of the repressive mark H3K9me3[18]. ATM also elicits MST2-dependent phosphorylation of H2B on S14 further leading to the recruitment of the chromatin compaction factor RCC1[19]. Beyond these modifications on chromatin, RNAPI transcriptional repression also depends on Treacle, a ribosomal DSB sensor, which allows TOPBP1 and MRN recruitment, and subsequent ATR activation, all essential to ensure full transcriptional shut-down[20,21].

### Remaining questions

While some reports suggested transcriptional repression to occur within entire γH2AX domains[11], hence spanning hundreds of kilobases around a single break, other suggested only a local effect, in the immediate (10kb) vicinity of the DSB[22,23]. Since cohesin is involved in DSB-induced gene silencing, deciphering how this transcriptional repression is organized in 3D is required to fully understand this arm of TC-DSBR.

## 2. Chromatin signaling and the bias for Homologous recombination
The use of tools to induce annotated DSBs in RNAPII and RNAPI transcription units also
revealed that both types of loci display a strong bias for resection and repair by HR (Fig. 2).

RNAPII-transcribed loci

Genome-wide studies using the DlvA system where multiple DSBs are induced throughout the genome by the AsiSI restriction enzyme indicated that DSBs located in RNAPII-bound loci exhibit much higher level of RAD51 recruitment than DSBs induced in intergenic loci[24], suggesting that TC-DSBs are channeled to HR repair. In agreement, transcription of the underlying locus before damage was found to be a key determinant for HR usage [24–26]. This bias for exacerbated resection at TC-DSBs depends on the histone mark H3K36me3, a modification associated to transcriptional elongation. H3K36me3 is recognized by LEDGF, which itself interacts with CtIP fostering resection at DSB induced in active genes [24,27]. Moreover, acetylated H4K16, linked to open, transcriptionally active chromatin, triggers the recruitment of the pro-resection factor BRCA1 by inhibiting the binding of the anti-resection factor 53BP1 to methylated H4K20[28]. Similarly H2AK15 acetylation by the Tip60 complex, by counteracting ubiquitylation of the same residue, impairs the recruitment of 53BP1 to H2AK15Ub[29]. Finally, low levels of H4K20me2 in open accessible chromatin decreases the ability of 53BP1 to be recruited at TC-DSB, therefore potentiating resection[30]. Altogether, all these studies demonstrated that the chromatin landscape established at RNAPII-transcribed loci prior to the appearance of any DNA lesion render these loci particularly prone to undergo resection and HR.

RNAPI-transcribed loci

Interestingly, multiple evidence indicate that the rDNA is also prone to undergo extensive
resection and HR repair. Work using dedicated CRISPR sgRNA to target the 45S ribosomal
DNA unit, the I-Ppol or AsiSI systems indicated that rDNA breaks tend to relocate at the
periphery of the nucleolus (see section 4), where they recruit RAD51, BRCA1 and other HR
factors[16,18,21,31,32]. Of interest, extended resection spanning >10kb, was found to occur at
DSB induced in rDNA[18].

*Remaining questions*

The bias for HR repair at TC-DSBs compared to intergenic loci only holds true for G2 cells, where the HR machinery is fully operational[24,25,33]. Notably in G1, TC-DSBs were shown to persist much longer than DSBs induced intergenic regions[33]. Whether TC-DSBs are repaired during G1 or later in the cell cycle, as well as the nature of this TC-DSBR pathway still remain to be fully understood. Surprisingly, oxidative DNA damage in active genes were found to be repaired by a Cockayne syndrome B-dependent homologous recombination in G0/G1 [34] and DSBs induced by CRISPR/Cas9 on the rDNA were reported to undergo HR even in G1[31], suggesting that HR could take place at TC-DSBs in some contexts in pre-replicative cells. Yet, the exact conditions in which this pathway can operate remains to be investigated given that this behavior at rDNA was not observed using other DSB induction systems[18,21]. Additionally, the contribution of NHEJ should be determined since it was shown to take place at rDNA break[16,35] and an RNA-templated NHEJ also reported to occur at TC-DSB[36].

**3. Which roles for RNAs?**

Recent work emphasized the role(s) of RNAs in TC-DSBR (Fig. 3).

*RNAPII-transcribed loci*

DNA:RNA Immuno-precipitation followed by High-throughput sequencing (DRIP-seq) revealed accrual of RNA:DNA hybrids at TC-DSBs[26,37]. Moreover, bidirectional transcription was observed at TC-DSB, using NET-seq[38] and live imaging[39]. It was proposed that RNA:DNA hybrids could (i) contribute to stabilize the single-strand DNA and regulate nucleofilament
assembly (e.g.[40]), (ii) stimulate HR by forming “DR-loop” (invasion of the RNA transcript into the donor DNA)[26], (iii) initiate and/or be the substrate for alternative repair pathways (e.g.[36,41]), (iv) act as recruitment platform for repair proteins, and/or be further processed to participate in DSB signaling (e.g.[42]). RNA:DNA hybrids must eventually be resolved to ensure Rad51 foci formation, proper HR and to avoid deleterious repair products such as translocations[37].

### RNAPI-transcribed loci

Induction of a DSB in rDNA also triggers *de novo* RNA production at the vicinity of the DSB that, surprisingly, is RNAPII-dependent. Such RNAs were found to give rise to diRNAs (damage induced small RNAs), likely by hybridizing to RNAPI-transcribed rRNAs precursors. These diRNAs may exert their function via loading into Argonaute (AGO) complexes[43].

### Remaining questions

The process responsible for producing these RNAs that are forming RNA:DNA hybrids at TC-DSBs still remains debated. Indeed, such RNAs could be produced thanks to the ability of DSB themselves to directly load RNAPII but could also arise from alterations to the transcription cycle occurring locally around TC-DSBs[44]. Additionally, by substituting to the double-stranded DNA substrate, RNA:DNA hybrids display a high potential to influence the repair process, and further studies are necessary to clearly establish whether they can serve as a template for repair, as primers for DNA synthesis, or/and as regulator of nucleofilament assembly and strand invasion[44]. Finally, how such hybrids are further processed, whether they give rise to single-stranded or/and double-stranded small RNAs, and what could be the function(s) of these processed RNA species remains to be addressed.

## 4. A tendency to move within the nucleus
Another very interesting feature of TC-DSBs is their ability to move and relocate within the nucleus (Fig. 4).

### RNAPII-transcribed loci

As previously mentioned (section2) DSB induced in RNAPII-transcribed loci tend to persist during the G1 cell cycle phase when compared to other DSBs induced in intergenic loci[33]. Importantly, in G1, TC-DSBs display the ability to coalesce within larger repair foci whose function is still unclear[33]. TC-DSBs clustering relies on ATM, the MRN complex, on the actin network and on the nuclear envelope-embedded LInker of Nucleoskeleton and Cytoskeleton (LINC) complex[33,45,46]. This clustering was proposed to be mediated by nuclear actin filaments, polymerized post DSB, in a manner that involves WASP and Arp2/3[45]. Fusion of DSB repair foci was also found to occur thanks to liquid-liquid phase separation mediated by 53BP1[47].

### RNAPII-transcribed loci

DSB induced in the rDNA also display high mobility and are rapidly relocated at the periphery of the nucleolus in "nucleolar caps" [16,18,20,31]. This mobilization depends on ATM and ATR activity[16,20] and precedes RAD51 assembly and repair by HR. Interestingly rDNA movement to the periphery of the nucleolus also depends on the actin network, and the LINC and MRN complexes[18]. Of note, nuclear envelope invaginations were also found to contact nucleolar caps formed after rDNA breaks raising the possibility that this specific nuclear envelope environment may contribute to rDNA repair, as previously reported for other types of persistent DSBs. Nucleolar segregation also depends on TOPBP1 recruitment at rDNA break[20], a protein further found to display liquid-liquid phase separation properties[48]. Altogether these data suggest that, as for the clustering of RNAPII-transcribed damaged loci, mechanism(s) that
involve the actin network, nuclear envelope components and phase separation ensure movement and proper localization of DSBs induced in RNAPI-transcribed loci.

*Remaining questions*

More work is required to understand the respective contribution of passive (through phase separation properties of repair factors, e.g. TOPBP1 or 53BP1) and active (through the involvement of nucleoskeleton and nuclear actin) processes in DSB mobility. Moreover, conflicting results exist regarding the cell cycle phase during which such mobility occurs, both for DSB induced in RNAPII-[33,45] and RNAPI-[18,21,31] transcribed loci. More importantly, the precise role of DSB mobility still needs to be fully understood. For rDNA DSB, it was suggested that this relocalization could allow for safe recombination repair, away from repetitive sequences, but this hypothesis still requires experimental validation. As for DSB clustering, several hypotheses can be considered: it could contribute to create a repair compartment and/or sequester persistent DSBs from the nucleus[49], or activate cell cycle checkpoints[47].

**Acknowledgments**

E.L. is recipient of a PhD fellowship from the Ligue Nationale Contre le Cancer. T.C. is an INSERM researcher. Funding in G.L.'s laboratory was provided by grants from the European Research Council (ERC-2014-CoG 647344), the Agence Nationale pour la Recherche (ANR-14-CE10-0002-01, ANR-18-CE12-0015) and the Ligue Nationale Contre le Cancer (LNCC). We apologize to colleagues whose work was not cited due to space constraints

**Conflict of Interest statement**

The authors declare that there are no conflicts of interest.
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**Figure 1: Local transcriptional repression at TC-DSBs in RNAPII- and RNAPI-transcribed loci**

DSBs in RNAPII- and RNAPI- transcribed loci elicit the local extinction of transcription in cis to DSB, through partially common mechanisms. Transcriptional repression at RNAPII-TC-DSB (left panel) relies on ATM, DNAPK, DYRK1B kinases activities and on the PARP1 poly-ADP polymerase activity. They directly and indirectly trigger (i) local modifications of the chromatin landscape, with a transition from “active” histone marks (H3K4me3, acetylated H3/H4) to “repressive” histone marks (H2AK119Ub, H3K27me3, H3K9me) and (ii) modifications of the RNAPII itself (parylation, ubiquitination) leading to pausing (through NELF-E recruitment) and degradation (via the proteasome). Transcriptional repression at RNAPI-TC-DSB (right panel) relies on ATM, DYRK1B and ATR kinases activities, that trigger HUSH-mediated -H3K9 trimethylation, and MST2-mediated H2B-S14 phosphorylation. To date, no direct DSB-induced modification on RNAPI has been reported. Both RNAPI- and RNAPII repression around TC-DSB involve the cohesin complex.

**Figure 2: TC-DSBs are prone to resection and HR repair.**

The chromatin landscape established prior and following DSB on RNAPII transcribed loci (left panel) render the DSB prone to resection. H3K36me3 serves as a docking platform for LEDGF which further recruits the resection factor CtIP. Meanwhile, Tip60 catalyzes H4K16ac and H2AK15ac which counteract H4K20me2 and H2AK15Ub, both required for efficient binding of the anti-resection factor 53BP1. At RNAPI- transcribed loci (right panel), such a chromatin signaling has not yet been identified but extended resection (>10kb) was reported to occur.
Both RNAPI- and RNAPII- TC-DSB repair pathways are regulated throughout the cell cycle: In G2, TC-DSBs are repaired by HR. In G1, TC-DSBR pathways are still unclear, but (i) coincide with delayed repair for RNAPII-TC-DSB, and (ii) may involve HR at RNAPI TC-DSBs.

**Figure 3: TC-DSBR involves RNAs.**

R-loops or RNA:DNA hybrids specifically accumulate in cis to DSB induced in RNAPII-transcribed loci (top panel). The mechanism that ensure their formation is yet unclear and may either involve *de novo* loading of RNAPII at DNA ends, or result from transcriptional repression. While these hybrids need to be eventually removed to ensure HR and avoid translocation, they may play a role at different steps during the repair process: (i) they likely regulate resection and the stability/assembly of RAD51 nucleofilament; (ii) they may contribute to alternative repair mechanisms, such as recently described RNA-templated repair pathway(s), and (iii) they could lead to double-strand RNAs (DDRAs, or diRNAs) proposed to be required for full DNA damage response. diRNAs also arise, in an RNAPII-dependent manner, from rDNA breaks, and are incorporated into AGO complexes (bottom panel).

**Figure 4: TC-DSBs are mobile**

Both DSBs induced in RNAPII- and RNAPI- transcribed loci (left and right panels respectively) display the intriguing ability to be mobile in the nucleus, in a manner that involves ATM kinase activity, the MRN complex, the actin network, and the LINC complex. Phase separation could also contribute to these mobility events (through 53BP1 for DSB in RNAPII-transcribed regions and TOPBP1 for DSB in rDNA). Mobility of TC-DSBs occurring in RNAPII-transcribed loci ensure clustering within larger repair foci, coinciding with delayed repair. Mobility of TC-DSBs in
RNAPI-transcribed loci ensures relocalization to the periphery of the nucleolus, in nucleolar caps. Interestingly, nucleolar caps contact nuclear envelope invaginations.
<figure><img src="image_3.png" /><figcaption>RNAPII-transcribed loci</figcaption></figure>

Deposition
of repressive histone marks

<figure><img src="image_4.png" /><figcaption>RNAPI-transcribed loci</figcaption></figure>

<figure><img src="image_5.png" /></figure>

Deposition
of repressive histone marks

DSB

<figure><img src="image_6.png" /></figure>
RNAPII-transcribed loci

resection

<figure><img src="image_7.png" /></figure>

Homologous recombination
(RNAPI &RNAPII)

<figure><img src="image_8.png" /></figure>

RNAPII-transcribed loci

extended resection

<figure><img src="image_9.png" /></figure>

Homologous recombination(?), NHEJ
(RNAPI)

Delayed repair
Alternative repair pathway?
(RNAPII)
<figure><img src="image_10.png" /><figcaption>RNAPI-transcribed loci</figcaption></figure>

<figure><img src="image_11.png" /></figure>
RNAPII-transcribed loci

<figure><img src="image_12.png" /></figure>

RNAPII-transcribed loci

<figure><img src="image_13.png" /></figure>