Transcription coupled repair at the interface between transcription elongation and mRNP biogenesis

Biochimica et Biophysica Acta 1829 (2013) 141–150

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Review

Transcription coupled repair at the interface between transcription elongation and mRNP biogenesis☆ Hélène Gaillard ⁎, Andrés Aguilera Centro Andaluz de Biologia Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Av. Américo Vespucio s/n, 41092 Sevilla, Spain

a r t i c l e

i n f o

Article history: Received 31 May 2012 Received in revised form 19 September 2012 Accepted 22 September 2012 Available online 6 October 2012 Keywords: Nucleotide excision repair Transcription coupled repair RNAPII transcription mRNP biogenesis CSB/Rad26 THO

a b s t r a c t During transcription, the nascent pre-mRNA associates with mRNA-binding proteins and undergoes a series of processing steps, resulting in export competent mRNA ribonucleoprotein complexes (mRNPs) that are transported into the cytoplasm. Throughout transcription elongation, RNA polymerases frequently deal with a number of obstacles that need to be removed for transcription resumption. One important type of hindrance consists of helix-distorting DNA lesions. Transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair, ensures a fast repair of such transcription-blocking lesions. While the nucleotide excision repair reaction is fairly well understood, its regulation and the way it deals with DNA transcription remains largely unknown. In this review, we update our current understanding of the factors involved in TC-NER and discuss their functional interplay with the processes of transcription elongation and mRNP biogenesis. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Nucleotide excision repair (NER) is a multiprotein-mediated process that removes a wide variety of helix-distorting DNA lesions such as UV-induced damage and bulky adducts. DNA repair by NER involves several steps, which consist of recognition of the DNA lesion, assembly of the repair complex, removal of the oligonucleotide containing the damage and filling of the gap by DNA synthesis using the opposite strand as template (reviewed in [1,2]). The major features of NER and its enzymology have been well documented and the core of the reaction reconstituted in vitro with purified proteins [3]. However, the precise mechanism of NER, its regulation, and the way it deals with DNA transcription remains largely unknown. NER can be divided into two sub-pathways, transcription-coupled repair (TC-NER), which removes DNA lesions that interfere with the progression of RNA polymerases (RNAP) through active genes, and global genome repair (GG-NER), which removes DNA lesions in the genome overall. TC-NER and GG-NER solely differ in the lesion recognition step while the core of the repair reaction is common to both subpathways. In GG-NER, specific proteins recognize the structural changes resulting from the DNA lesion (e.g. helix distortions and changes in H-bonding). In TC-NER, the elongating RNAP stalls at the DNA lesion

☆ This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation. ⁎ Corresponding author. Tel.: +34 954 46 77 28; fax: +34 954 461 664. E-mail address: [email protected] (H. Gaillard). 1874-9399/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagrm.2012.09.008

and promotes the recruitment of NER factors, thereby increasing the efficiency of the repair reaction. In eukaryotes, TC-NER depends on RNAPII-driven transcription, although reports have shown that TCNER also occurs in RNAPI transcribed rDNA genes in yeast [4,5]. The recent demonstration that transcription arrest by the bacteriophage T7 polymerase also triggers TC-NER [6] highlights the universality of the phenomena. Several additional factors are involved in TC-NER, for many of which the precise function remains unclear. The proteins involved in TC-NER, GG-NER, or NER in eukaryotes are listed in Table 1. DNA repair by NER can be analyzed in vivo by monitoring the removal of UV-induced DNA damages at specific loci [7]. Cells are irradiated with a defined dose of UV light to induce DNA lesions and grown further to allow repair. The most abundant type of DNA lesion generated by UV light are cyclobutane pyrimidine dimers (CPDs), which can be cleaved in vitro by the DNA nicking activity of a repair enzyme called T4 endonuclease V. Strand-specific repair at a given locus is determined by denaturating Southern analysis using single-stranded probes. Preferential DNA repair of the transcribed strand (TS) over the non-transcribed strand (NTS) of active genes was described more than two decades ago both in human cells and in bacteria [8,9]. The findings that TC-NER also takes place in the baker's yeast Saccharomyces cerevisiae came shortly after [10,11]. In humans, defective TC-NER is mainly associated with two autosomal-recessive disorders called Cockayne (CS) and UV-sensitive syndromes (UVsS). CS is characterized by UV-sensitivity, premature aging and progressive neurodevelopmental abnormalities, and UVsS by photosensitivity (reviewed in [12,13]). During the transcription process, active genes are in a ‘loose’ conformation and might therefore be more accessible to DNA repair enzymes.

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Although open chromatin structure generally improves the accessibility of repair enzymes (reviewed in [14]), the facts that TC-NER strictly depends on the presence of a transcribing RNAP, is restricted to the strand being transcribed, and is not restricted to chromatin-packed genomes, as it also takes place in bacteria, argue for an active coupling of transcription and repair. This review aims to update our current knowledge on how NER is coupled to transcription and to revise the function of the factors that play a role in TC-NER. 2. Coupling transcription with DNA repair in bacteria In Escherichia coli, the mechanism underlying TC-NER is fairly well understood (reviewed in [15,16]). Coupling between transcription and repair is mediated by one single protein called Mfd, which releases arrested RNAPs and recruits the NER enzyme UvrA [17] (Fig. 1). The remainder of the NER repair enzymes (UvrB, UvrC, UvrD, DNA polymerase I, and DNA ligase) are then recruited to complete the repair reaction. Mfd is an ATP-dependent translocase that interacts with paused transcription complexes by interacting with both the β subunit of RNAP and the DNA immediately upstream of the stalled RNAP [18–20]. Mfd translocase activity can reactivate transcription elongation by pushing the paused polymerase to realign the RNA transcript in the active site. In the case of an arrested transcription complex, as it occurs at a DNA damage, Mfd activity destabilizes the stalled transcription complex, leading to release of the RNAP and nascent mRNA from the damaged DNA. In addition, Mfd interacts with the NER protein UvrA, which works in DNA damage recognition, the earliest step of the repair pathway. Recently, a detailed analysis of TC-NER in different Mfd and UvrA mutants demonstrated that the interaction between Mfd and UvrA is essential for Mfd to increase the repair rate on the TS [21]. The results of this study suggest that Mfd increases the repair rate by accelerating the DNA damage recognition step. Nevertheless, other factors stimulate UvrA recruitment to damaged sites independent of transcription [22,23]. The development of a single-molecule assay to monitor Mfd interactions with single stalled RNAP molecules demonstrated that the intermediate formed by Mfd, the arrested RNAP, and the DNA is reliably long-lived and, therefore, could act as a marker for sites of DNA damage [24]. Further development of the system, including the addition of subsequent NER proteins such as UvrA, will provide useful information to improve our understanding of the TC-NER mechanism in bacteria. In addition, the essential transcription factor NusA has been proposed to mediate an Mfd-independent TC-NER sub-pathway [25]. Evidence supporting this hypothesis includes the sensitivity of nusA, uvrA, and RNAP mutants to the damaging agent nitrofurazone (NFZ), the ability of chemically induced N 2-furfuryl-dG (a structural analog of the principal lesion generated by NFZ) to block RNAP transcription in vitro, as well as physical and genetic interactions between NusA and UvrA. NusA is known to participate in transcription termination, increasing the efficiency of intrinsic termination, affecting Rhodependent termination, and mediating anti-termination of bacteriophage λ. Interestingly, however, it also moderates the elongation rate of RNAP (reviewed in [26]). 3. Coupling transcription with DNA repair in eukaryotes 3.1. The CSB/Rad26 and CSA factors In eukaryotes, DNA damage is recognized by specialized protein complexes in GG-NER, which are dispensable for the TC-NER reaction [27,28]. These protein complexes are XPC-HR23B and DDB1-XPE/DDB2 in human and Rad7-Rad16 in yeast. This differs from E. coli UvrA, which acts both in GG-NER and in TC-NER [29]. As in bacteria, other factors appear to be required specifically for TC-NER in addition to the elongating RNAP both in yeast and in humans. The first proteins that were found necessary for TC-NER are the Cockayne's syndrome proteins A and B (CSA and CSB, respectively) [30,31]. The requirement for CSB to assemble

a functional TC-NER complex has been demonstrated both in vitro [32] and in vivo as CSB recruits other factors including CSA, the transcription initiation/NER factor TFIIH, the earliest indispensable NER factor XPA, and the NER endonuclease XPF-ERCC1 [33]. In cooperation with CSB, CSA is required to recruit the tetratricopeptide-containing protein XAB2 to DNA lesion-stalled RNAPII [33]. XAB2 was originally identified as an interaction partner of XPA – the earliest indispensable NER factors – in a two-hybrid screening [34]. Microinjection of anti-XAB2 antibodies led to the inhibition of NER in XPC cells, in which TC-NER is the only form of NER, suggesting that XAB2 plays a role in TC-NER. Additionally, XAB2 appears to form part of a complex involved in pre-mRNA splicing in HeLa cells [35]. Since tetratricopeptide motives are thought to mediate protein–protein interactions, XAB2 has been proposed to work as a scaffold for the formation of TC-NER protein complexes. However, further work is required to clarify the function of XAB2 in TC-NER. CSB also recruits the histone acetyltransferase complex p300 to the site of damage [33]. p300 is also associated with other repair enzymes including the GG-NER damage recognition proteins DDB1 and XPE/ DDB2, thymine-glycol DNA glycosylase, and DNA damage checkpoint kinase ATR, suggesting that p300 may improve DNA accessibility in different DNA repair pathways. The non-histone nucleosome binding protein HMGN1, which is also implicated in TC-NER [33,36], might as well contribute to increasing DNA accessibility to the repair enzymes. Because TC-NER takes place in active genes, the chromatin structure might not be the limiting factor of the reaction, in contrast to GG-NER, where chromatin remodeling is required in the context of condensed chromatin (reviewed in [37]). The S. cerevisiae homologs of CSA and CSB, Rad28 and Rad26, were identified based on sequence similarity. Deletion of the RAD26 gene also results in TC-NER defects [38], whereas Rad28 null mutants are TC-NER proficient [39]. Although Rad26 is required for TC-NER, residual TC-NER activity remains in rad26 null mutant, suggesting the involvement of other factors [40]. Repair analysis of several genes at nucleotide resolution indicates that Rad26 requirement is limited to the transcription elongation region, but dispensable for efficient repair of the promoter, a few nucleotides immediately downstream of initiation, and termination regions [41,42]. These observations led to the proposal that Rad26's role might reside in the recruitment of TFIIH to elongating RNAPII stalled at DNA damage. Although a Rad26-dependent recruitment of TFIIH has not been demonstrated in yeast to date, the fact that CSB does recruit TFIIH to stalled polymerase [33] supports this hypothesis. This model implies that CSB/Rad26 would either be recruited once RNAPII stalls at a DNA lesion or go along with the elongating RNAPII independently of the presence of damages. The findings that both Rad26 and CSB play a role in transcription elongation in the absence of exogenous DNA damage [43–45] and that Rad26 associates with the coding sequence of genes in a transcription-dependent but DNA damage-independent manner [46] rather support the second possibility. However, Rad26 was shown to transiently localize to active genes upon DNA damage, while Ser5-P RNAPII was transiently lost [47]. The early role of Rad26 in TC-NER has been further sustained by the finding that it is phosphorylated in a Mec1-dependent manner upon UV damage and that the phosphorylation site is required for TC-NER [48]. In addition to its implication in TC-NER, Rad26 plays a role in the repair of repressed genes, especially in the core regions of well-positioned nucleosomes [49]. These results are in line with the previous report showing that over-expression of Rad26 increases both TS and NTS repair of active genes, and partially bypasses the requirement for Rad7 in GGNER [50]. The recent finding that Rad26 does not recognize DNA lesions in the absence of active transcription [46] suggests that the involvement of Rad26 in the repair of repressed genes might rely on non-productive RNAPII-driven transcription, a widespread phenomenon in eukaryotes (reviewed in [51]). Interestingly, a non-coding RNA covering the GAL110 locus is transcribed in repressive conditions [52]. GAL1-10 is precisely one of the loci where Rad26 contributes to DNA repair in the absence of productive transcription [49].

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Table 1 Proteins involved in NER.

Step

DNA

Pathway

GG-NER

damage recognition TC-NER NER

Additional

TC-NER

factors

S. cerevisiae

Human

Function/activity

Rad7-Rad16

−

Damaged DNA binding

Rad4-Rad23

XPC-HR23B

−

DDB1-DDB2/XPE

RNAPI,

RNAPI,

RNAPII

RNAPII

Rad4-Rad23

XPC-RAD23B

Damaged DNA binding, assembly platform Damaged DNA binding, XPC recruitment RNA polymerase DNA damage binding TCR coupling factor,

Rad26

CSB

Rad28

CSA

E3 ubiquitin-ligase

−

UVSSA-USP7

Protein deubiquitylation

Rpb9

RPB9

Non-essential RNAPII subunit

Kin28

CDK7

Hpr1, Tho2

THOC-1, THOC-2

Sub2

UAP56

Thp1

PCID2

PAF

PAF/Paf1

Transcription elongation factor

Ccr4-Not

Ccr4-Not

Transcription factor, deadenylase

CFI

CstF

transcription elongation

C-terminal domain kinase, TFIIH subunit mRNP biogenesis, THO subunits mRNP biogenesis, TREX subunit mRNP biogenesis, THSC/TREX-2 subunit

Cleavage factor, CstF has been implicated in TC-NER in chicken DT40 cells Syf1

XAB2

−

BRCA1

−

HMGN1

XPA-binding protein, transcription factor Ubiquitin-ligase activity in BRCA1-BARD1 heterodimer Non-histone chromosomal protein, chromatin relaxation

NER

−

p300

Histone acetyltransferase DNA damage binding, lesion

Rad14

XPA

verification, complex

Rfa1, 2, 3

RPA p70, p32, p14

ssDNA binding

Ssl2/Rad25

XPB

Rad3

XPD

Rad2

XPG

stabilization Open complex formation

NER

and incision reaction

Gap filling and ligation

NER

3' to 5' helicase, TFIIH subunit 5' to 3' helicase, TFIIH subunit Endonuclease (3' incision), open complex stabilization

Rad1-Rad10

XPF-ERCC1

Endonuclease (5' incision)

PCNA

PCNA

DNA replication sliding clamp

Rfc1/Cdc44

RFC1

PCNA loading

DNA pol ∂, ε

DNA pol ∂, ε

DNA replication

Cdc9

DNA ligase I

DNA ligation

–, homologous protein is absent or unknown; gray, genetic homolog that does not share function; brown, genetic and/or functional homolog which has not been analyzed yet in NER.

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suggest that this role includes the stabilization of CSB by targeting the deubiquitinating enzyme USP7 to lesion-stalled RNAPII complexes.

RNAP

3.3. Function and regulation of the CSB protein

DNA lesion Mfd

RNAP

UvrA

RNAP

RNAP

UvrB

DNA cleavage Oligonucleotide displacement Patch synthesis

Fig. 1. Coupling of DNA repair and transcription in E. coli. In prokaryotes, translation and transcription occur simultaneously. Stalling of elongating RNAP at a transcription-blocking DNA lesion leads to the recruitment of Mfd, which in turn recruits NER factor UvrA. Mfddependent DNA translocation removes RNAP from the damaged DNA. UvrB is recruited to the DNA lesion leading to the formation of a pre-incision complex. The following DNA repair steps are common to GG-NER and involve cleavage of the DNA, removal of the oligonucleotide containing the damage, gap filling by DNA synthesis, and ligation.

3.2. The UVSSA factor UVsS is a human DNA repair-deficiency disorder characterized by skin photosensitivity. Cells from UVsS patient are defective in TC-NER of UV lesions but show normal GG-NER [53]. Recently, mutations in the UVSSA (UV-stimulated scaffold protein A) gene were found to be the cause of UVsS [54–56]. UVSSA forms a stable complex with the ubiquitin-specific protease USP7, and depletion of either of proteins leads to decrease in UV survival, in RNA synthesis recovery after UV and in CSB stability [55,56]. Upon UV damage, UVSSA physically interacts with CSA, CSB, TFIIH, XAB2, HMGN1, and RNAPII, indicating that it is recruited to stalled polymerase during the initiation of the TC-NER reaction [54–56]. Furthermore, green fluorescent protein-tagged UVSSA accumulated at local UV damage with similar kinetics as CSB in living cells [56]. Together, these studies indicate that UVSSA plays a role in TC-NER at UV damages and

CSB/Rad26 belong to the SWI2/SNF2 helicase superfamily and have been proposed as putative eukaryotic transcription-repair coupling factor candidates. However, while the action of the Mfd translocase leads to the release of RNAP from the damaged strand in E. coli [17], CSB does not disassemble RNAPII [57]. Because CSB shows intrinsic ATP-dependent chromatin remodeling activities in vitro [58,59], a role for CSB to increase the accessibility of DNA lesion to the repair machinery has been postulated, although this is unlikely to be the only role of CSB, as it acts exclusively on the transcribed strand of active genes. CSB is normally phosphorylated in non-irradiated cells and its dephosphorylation in response to UV results in increased ATPase activity [60]. The analysis of mutants carrying different mutations in the ATPase domain of CSB showed enhanced UV-sensitivity in clonogenic survival assays, increased percentage of apoptotic cells after UV-exposure as well as impaired resumption of transcription, although variations in the strength of these phenotypes were observed depending on the mutation site [61]. However, an ATPase-deficient mutant partially restores CSB activity in vivo [62] and CSB appears to remodel chromatin in the absence of DNA damage [63]. Interestingly, the N-terminal region of CSB negatively regulates its binding to chromatin. ATP hydrolysis by CSB is required to alleviate this effect upon UV-irradiation [64], suggesting that CSB recruitment to lesion-stalled RNAP involves major conformational changes. In mammals, the CSA protein is strictly required for TC-NER and its absence gives rise to Cockayne syndrome to the same extent as does the absence of CSB. CSA is an E3 ubiquitin ligase that is recruited to the site of damage by CSB. CSA is known to ubiquitylate CSB, targeting it to degradation by the proteasome pathway [65]. The recent discovery that the C-terminal part of CSB contains a ubiquitin-binding domain (UBD) that is absolutely required for TC-NER [66] led to the hypothesis that CSA is required for the ubiquitylation of the ubiquitylated partner of CSB, whose identity still remains to be determined. When the predicted UBD is deleted in CSBdel, TC-NER complexes seem to assemble as determined by co-IP experiments but no DNA repair occurs. Instead, repair is restored when CSB's UBD is replaced by a heterologuous UBA2-domain. Therefore, these results suggest that ubiquitylation of a still unknown protein by CSA and binding of CSB to this ubiquitylated partner might be required to ‘license’ the DNA repair reaction. As the newly identified TC-NER factor UVSSA is normally ubiquitylated and physically interacts with CSA, CSB, and RNAPII [54–56], it represents a plausible candidate for CSB's ubiquitylated partner. Furthermore, the deubiquitylating enzyme USP7 stably associates with UVSSA and has been proposed to regulate the stability of CSB [55,56]. Altogether, these results suggest a model in which the early steps of TC-NER at a stalled RNAPII are regulated by series of ubiquitylation and deubiquitylation steps, which might possibly determine the outcome of the repair reaction. Notably, the yeast CSB homolog Rad26 lacks a ubiquitin-binding domain, the CSA homolog Rad28 is not required for TCR [39], and no UVSSA homolog has been identified in yeast, indicating that the TC-NER mechanism is not fully conserved in yeast and humans. Another protein that polyubiquitinylates CSB in response to UV damage is BRCA1, one of the breast and ovarian cancer susceptibility genes [67]. This activity occurs independently of CSA, suggesting that the two proteins might have partially redundant activities. Although both BRCA1- and CSA-deficient cells show defects in the removal of oxidative base damage and CPD from the transcribed DNA strand ([67,68] and references therein), biologically distinct types of lesion might require the action of different factors. Additional studies are needed to gain understanding of the specific roles played by BRCA1 and CSA in mammalian TC-NER.

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4. TC-NER and the interface between transcription elongation and mRNP export 4.1. The THO, TREX, and THSC/TREX-2 complexes During the transcription process, the nascent pre-mRNA associates with mRNA-binding proteins and undergoes a series of processing steps, resulting in export-competent mRNA particles (mRNPs) that are transported into the cytoplasm. Over the past several years it became clear that the different RNA processing steps (including 5′-end capping, splicing, and 3′-end cleavage), mRNP export, and transcription are connected to each other, and that surveillance mechanisms ensure that these processes occur in a coordinated manner (reviewed in [69–71]). The THO complex is a conserved five-protein complex composed of stoichiometric amounts of Tho2, Hpr1, Mft1, Thp2, and Tex1 that function at the interface between transcription elongation and mRNP biogenesis (reviewed in [72,73]). A fraction of THO associates with the mRNA export proteins Sub2/UAP56 and Yra1/Aly to form a larger complex termed TREX in yeast and humans [74]. Many phenotypes of THO and TREX are shared by the THSC/TREX-2 complex which consists of the Thp1, Sac3, Sus1, Sem1, and Cdc31 proteins and function in mRNA export by docking the mRNP to specific nucleoporins at the nuclear pore entry (reviewed in [75,76]). CPD repair analyses revealed that mutants of THO (hpr1 and tho2), TREX (sub2), and THSC/TREX-2 (thp1) are impaired in TC-NER [77,78]. These studies suggest that TC-NER deficient RNAPII complexes remain stalled at transcription-blocking DNA lesion in THO, Sub2 and THSC/TREX-2 mutants. On the mRNA export route, THO works in immediate proximity to the ternary complex formed by the polymerase holoenzyme, template DNA and nascent mRNA, whereas Sub2 and THSC/TREX-2 work downstream in the mRNP biogenesis and export route. Ribozyme-mediated cleavage of the nascent mRNA, which is the only known physical link between these complexes, is not sufficient to rescue TC-NER [78]. Thus, unidentified bridging factors might link mRNA export factors to the elongating RNAPII machinery. Absence or alteration of these factors in export mutants might result in an incomplete or modified transcription apparatus, which in turn would be incompetent to promote TC-NER, possibly as a consequence of its impaired transcription processivity. Syf1, the yeast homolog of XAB2, is part of the Prp19 complex and was recently identified as a new transcription elongation factor [79]. Interestingly, Syf1 interacts genetically and physically with THO and is recruited to transcriptionally active genes [79]. These results and the implication of XAB2 in TC-NER [34], designate the Prp19 complex a possible bridging factor between mRNA export factors and the elongating RNAPII machinery. In this line, it would be compelling to analyze whether mRNP biogenesis and export mutants lead to structural changes in elongating RNAPII or in factors associated with it.

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Noteworthily, recent reports indicate that Ccr4-Not interacts with the mRNP surveillance and export machineries [83–85] and PAF has functions in mRNA 3′-end processing [86]. The termination factor CstF appears to play a role in TC-NER, as its depletion reduces the preferential repair of the TS over the NTS in an exogenously irradiated reporter gene in chicken DT40 cells [87]. Although the mRNP biogenesis complexes involved in TC-NER are conserved from yeast to higher eukaryotes, it remains to be determined whether they function in TC-NER in mammalian systems. In THO mutants, R-loops consisting of RNA:DNA hybrids and the displaced ssDNA accumulate behind elongating RNAPII and account for the hyper-recombination phenotype of these cells [88]. R-loop accumulation also triggers genomic instability in mutants of THSC/ TREX-2, PAF, Ccr4-Not, and in mutants of cleavage factor I (CFI), the yeast homolog of CstF [89–91]. G-rich homopurine–homopyrimidine stretches are predicted to form unusually stable R-loops, which in turn are believed to be responsible for transcriptional blockage of such sequences [92]. Thus, it is conceivable that the presence of aberrant R-loops in transcribed genes might represent a direct impediment to the TC-NER reaction or trigger ‘gratuitous’ TC-NER in the absence of DNA damage. However, the finding that cleavage of the nascent mRNA does not suppress the TC-NER defects of THO mutants and that a THO allele that does not accumulate R-loops (hpr1-101; [93]) is as sensitive to UV in the absence of GG-NER as a full deletion mutant [78] does not support this hypothesis. Yet these results do not exclude that the presence of R-loops might impact TC-NER efficiency, or that R-loop accumulation and TC-NER deficiency might be independent consequences of mRNP biogenesis deficiencies. Further work will be required to dissect the causality and relationship between the two processes. The nuclear envelope marks a fundamental difference between eukaryotic and prokaryotic cells. The obligatory passage of eukaryotic mRNA through the nuclear envelope to reach protein translation machineries is tightly controlled by mRNA export systems. TC-NER has to cope with mRNA processing and export in eukaryotes, possibly explaining why it cannot take advantage of one main transcriptionrepair coupling factor as found in prokaryotic cells. The existence of a surveillance mechanism that might sense mRNP biogenesis and export deficiencies like those associated with THO, Sub2 and THSC/TREX-2 mutations has been proposed [78]. Such a surveillance mechanism could sense the structural problems arising from mRNP biogenesis deficiencies, such as nuclear retention of mRNPs, co-transcriptional R-loop accumulation, or RNAPII blockage for example, and act on the elongating RNAPII, possibly via some bridging factors, in such a way that its proficiency for TC-NER is significantly reduced. Alternatively, it is conceivable that the presence of DNA lesions on the TS might modulate downstream RNA processing and that the repair of these lesions by TC-NER requires plasticity in the downstream mRNP biogenesis and export machineries.

4.2. Other factors linking mRNP biogenesis and TC-NER 5. TC-NER and transcription elongation Interestingly, in addition to THO, Sub2, and THSC/TREX-2, other factors working at the interface between transcription and mRNP biogenesis have an impact on TC-NER, as shown in a study in which mutants showing sensitivity to the transcription elongation inhibitor mycophenolic acid were tested for defects in TC-NER by UV sensitivity in a GG-NER deficient strain (rad7) and CPD repair analysis in the constitutively transcribed RPB2 gene [80]. This work revealed a role for the two multi-protein complexes PAF and Ccr4-Not in TC-NER. The PAF/Paf1 complex is conserved among eukaryotes and regulates RNAPII transcription at multiple levels including initiation, elongation, termination as well as chromatin modifications (reviewed in [81]). The Ccr4-Not complex is a global regulator of gene expression that is conserved from yeast to humans. It is a large complex with multiple roles in gene expression, including regulation of transcription initiation, elongation, and cytoplasmic mRNA degradation (reviewed in [82]).

The process of transcription occurs in three phases: initiation, elongation, and termination. While the RNAP is involved all along the process of transcription, many transcription factors are specifically required for one of the transcription steps. The CSB/Rad26 TC-NER factor plays a role in transcription elongation in the absence of DNA damage [43–45]. Notably, several other transcription elongation factors have been implicated in yeast TC-NER, including Rpb9 and Kin28, which are subunits of RNAPII and of the general transcription factor TFIIH, respectively. Rpb9 is required for growth at extreme temperatures [94], accurate start site selection [95], transcription fidelity control [96], transcription elongation [97,98], and TC-NER [99]. It is composed of three distinct domains: the N-terminal zinc ribbon domain (Zn1), the C-terminal zinc ribbon domain (Zn2), and the central linker. While both the Zn1 and Zn2 domains are required for transcription elongation [100], only Zn1 was shown to be

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essential for TC-NER [101] whereas Zn2 is required for accurate start site selection [102]. TFIIH functions both in RNAPII transcription initiation and in NER. Kin28 is involved in the transition from transcription initiation to elongation by phosphorylation of the carboxy-terminal domain (CTD) of the largest RNAPII complex subunit (at Ser5 and Ser7 residues; [103] and references therein). Recent data using a specific chemical inhibitor of Kin28 kinase activity suggest that CTD phosphorylation by Kin28 is rather required for proper 5′-capping of the nascent transcript than for transcription elongation itself [104,105]. A thermo-sensitive KIN28 allele displayed a specific TC-NER defect at the non-permissive temperature (and no GG-NER defects) [106]. Although this TC-NER defect might rely on the poor RNAPII transcription observed in the mutant strain, the fact that GG-NER was not affected indicates that TFIIH was still proficient for repair in the conditions used. In mammals, a role in TC-NER was proposed for the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as its depletion seems to specifically alter the TS repair of the DHFR gene using a newly developed assay based on strand-specific PCR amplification [107]. The finding that DNA-PKcs interacts with the Cyclin T2 subunit of the p-TEFb elongation factor led the authors to propose that the implication of DNA-PKcs in TC-NER may rely on its association with p-TEFb. DNA-PK is a member of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family with serine/threonine kinase activity that plays a major role in non-homologous end joining repair ([108] and references therein). Since DNA-PK mutant cells are sensitive to UV and impaired in NER [109], further studies would be required to demonstrate the role played by DNA-PK in NER and in TC-NER. Fast repair by TC-NER only takes place on transcription-blocking lesions and strictly requires the presence of an elongating RNAP. Moreover, most factors implicated in TC-NER participate in transcription elongation in the absence of DNA damage, including the factors working at the interface between transcription elongation and RNA export such as THO/ TREX, THSC/TREX-2, PAF/Paf1, or Ccr4-Not. These features suggest that efficient mRNA synthesis might be a pre-requisite for TC-NER proficiency. However, yeast transcription-elongation mutants of the Spt4-Spt5 and the Bur1-Bur2 elongation complexes (spt4 and bur2) do not show defects in TC-NER [110] nor increase in UV sensitivity in rad7 mutants in which GG-NER is nearly absent [80], respectively. On the contrary, spt4 suppresses and Bur kinase inactivation partially alleviates the TC-NER defects of rad26 [110,111], indicating that reduced RNAPII elongation can even act positively on TC-NER. Although the precise mechanism of suppression is not known, recent results suggest that the effect is mediated by the Spt5 protein, which is stabilized by Spt4 and phosphorylated by the Bur kinase [111]. Along the same line, the RNAPII subunit Rpb4 does not show TC-NER defects but on the contrary its removal appears to suppress the TC-NER defects of rad26 cells in the stationary phase [99]. Furthermore, analysis of TC-NER in a TFIIE yeast mutant in which transcription is significantly reduced demonstrated that TC-NER occurs even at low levels of RNAPII transcription [112]. Thus, it appears that the TC-NER phenotype is not only linked to elongation efficiency, but that the activity of specific factors is required for efficient TC-NER. It is noteworthy that TC-NER also takes place in RNAPI transcribed rRNA genes in yeast [4,5] and CSB associates with RNAPI and promotes rRNA transcription in humans [113–115]. Interestingly, both the PAF/ Paf1 complex and TFIIH work as elongation factors of RNAPI transcription [116,117]. Therefore, a systematic analysis of the differences and similarities between the two kinds of transcription could be extremely useful in dissecting the key features of elongating RNAP that are required for TC-NER. 6. The fate of a RNAP stalled at DNA damage The key player in TC-NER is certainly the elongating RNAP, since it is RNAP stalling at damaged DNA bases that efficiently triggers the repair reaction, which consists of the very same steps as in GG-NER. Because

the CPD responsible for stalling of the elongating RNAPII is located within the enzyme active site, it is not directly accessible to NER factors [118]. The mechanism by which NER factors are recruited to the DNA damage remains largely unknown. However, it is generally accepted that one of the first steps in TC-NER is to dispose of the stalled RNAPII at the site of damage, either by RNAPII removal, RNAPII retrograde translocation, or RNAPII DNA damage bypass (Fig. 2). Transcriptional arrest induced by DNA damage results in ubiquitination and proteasome-mediated degradation of RNAPII [119,120]. In humans, UV-induced ubiquitination of RNAPII is deficient in fibroblasts from individuals suffering either form of CS (CSA and CSB) and can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively [119]. In yeast, RNAPII ubiquitylation in UV irradiated cells depends on the Def1 protein, which forms a complex with Rad26 [121]. Def1 is directly required for RNAPII ubiquitylation in a reconstituted system as it recruits the ubiquitylation machinery and its binding to RNAPII is DNA damage specific [122]. Def1 deletion enhances UV sensitivity of GG-NER-deficient (rad16) or NER-deficient (rad14) strains, but does not affect the repair efficiency of the constitutively expressed RPB2 gene. These phenotypes are consistent with a role for Def1 in the DNA damage response independent of the repair reaction itself. The finding that def1 cells are unable to degrade RNAPII in response to DNA damage while the absence of Rad26 gives rise to an increased breakdown of RNAPII led to a model in which a regulatory mechanism decides between displacement and degradation of a stalled RNAPII molecule (reviewed in [123]). More recently, Rpb9 has been shown to promote ubiquitylation and degradation of Rpb1, the largest subunit of RNAPII, in response to UV irradiation [124]. This activity requires the Zn2 domain, which is dispensable for the transcription elongation and TC-NER functions of Rpb9. In chicken DT40 cells, the CstF termination factor is also required for UV-induced proteosomal degradation of RNAPII [87]. Together, these data suggest that RNAPII degradation belongs to the cellular response to DNA damage and occurs in coordination with the TC-NER process. An alternative to RNAPII degradation would be backtracking of the polymerase along the DNA template to allow DNA repair and resuming of transcription once the DNA lesion has been removed. Several lines of evidence support this possibility, as damage-stalled RNAPII appears to remain on the DNA template during the repair reaction in mammalian cells [33] and does not shield the lesion from accessibility to repair enzymes in vitro [125–127]. During backtracking, the RNAPII shifts backward along the DNA template. Subsequent transcription restart depends upon cleavage of the extruded mRNA to reposition the 3′-end of the RNA to the active site of the RNAPII [128]. TFIIS is a transcription elongation factor that helps RNAPII bypass transcription arrest sites. Mechanistically, it stimulates an intrinsic mRNA cleavage activity of the RNAPII [129,130], resulting in the reverse translocation of the polymerase until the mRNA realigns with the DNA template strand. This allows the polymerase to resume transcription, possibly bypassing the original arrest site. In vitro studies showed that TFIIS elicits a cryptic nuclease activity in a CPD stalled RNAPII polymerase, thus allowing it to back up away from the lesion [126,131]. This ability might confer TFIIS a capacity to contribute to TC-NER. In vivo, chromatin IP showed that TFIIS is recruited to active genes upon UV in a CSA-dependent manner [33]. However, direct analysis of UV-sensitivity, CPD repair and postUV transcription restart in cells depleted of TFIIS showed that TFIIS is not required for TC-NER in vivo, but might rather play a role in transcription recovery after UV damage [132,133]. Interestingly, TFIIS is not required for TC-NER in yeast either [134]. Noteworthy, a recent report showed that the Ccr4-Not complex, a global regulator of gene expression, directly interacts with elongating RNAPII complexes in vivo and stimulates transcription elongation of arrested polymerases in vitro [135]. Reactivation of backtracked RNAPII occurs by a different mechanism as described for TFIIS, as Ccr4-Not does not stimulate the nucleolytic activity of RNAPII and elongation restart depends on nascent transcript length. Whether this feature of Ccr4-Not is linked to its requirement for efficient TC-NER remains to be determined.

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XPA RNAPII

Fig. 2. The fate of an RNAPII stalled at a transcription-blocking DNA lesion. In eukaryotes, the transcription process is tightly coupled with mRNA processing and the formation of export-competent mRNPs. Various protein complexes working at the interface between elongation and mRNA export are required for efficient TC-NER, including THO, Sub2/UAP56, THSC/TREX-2, PAF, and Ccr4-Not. Stalling of elongating RNAPII at an obstructive DNA lesion triggers the recruitment of NER factors to ensure rapid removal of the DNA damage and resumption of transcription. Three main mechanisms, which are not necessarily mutually exclusive, are proposed to expose the DNA damage to the repair enzymes. i) RNAPII removal via proteosomal degradation, which depends on the Def1 protein in yeast. The fate of CSB/Rad26 is currently unknown. The exposed DNA lesion gets repaired by NER and transcription re-initiates. ii) RNAPII retrograde translocation along the template DNA exposes the DNA damage. CSA and the early NER repair factors TFIIH and XPA are recruited by CSB/Rad26. Once repair is complete, transcription resumes. iii) RNAPII lesion bypass, which might occur with concomitant recruitment of NER enzymes or not. CSB/Rad26 might favor lesion bypass. Transcription continues and the DNA lesion gets repaired.

A third possibility to remedy the problem caused by an elongating RNAP stalled at DNA damage could be to transcribe across the DNA lesion. Prokaryotic RNAP were shown to bypass non-bulky DNA lesions in an error-prone process known as transcriptional mutagenesis (reviewed in [136]). Characterization of the transcripts resulting from in vivo bypass of bulky DNA damage such as cyclo-dA and CPD by human RNAPII demonstrated that transcriptional mutagenesis occurs in these conditions [137]. Interestingly, this study suggested that bulky lesion bypass by human RNAPII might not always be mutagenic, as wild-type mRNAs were recovered in addition to mutated mRNA. Recently, yeast RNAPII was shown to possess intrinsic translesion synthesis properties allowing error-free bypass of a CPD lesion [138]. It is conceivable that CPD lesion bypass occurs in the context of TC-NER, since analysis of a CSB-deficient cell line provided evidence that CSB increases the probability of lesion bypass in vivo [137] and CSB promotes the addition of one nucleotide in vitro [43]. The idea that CSB/Rad26 might enhance the intrinsic translesion synthesis properties of RNAPII is consistent with the observation that CSB prevents both backtracking and the action of TFIIS on RNAPII arrested at CPD and cisplatin-induced DNA cross-links [32,43], and that Rad26 promotes lesion bypass at DNA damages induced by the alkylating agent methyl methansulfonate independently of both NER and base excision repair [139]. Alternatively, CPD bypass might occur independently of the presence of repair proteins. 7. Conclusions and perspectives The mechanism by which NER factors are efficiently recruited to the transcription-blocking DNA damage in TC-NER remains largely unknown. Structural analysis revealed that yeast RNAPII does not experience conformational changes upon stalling at a CPD lesion [118], suggesting that NER factor recruitment is not mediated by allosteric changes. However, since numerous modifications of the CTD of RNAPII are involved in the regulation of the transcription process in eukaryotes, specific modifications of RNAPII's CTD might be involved in signaling the DNA damage to NER enzymes. By analogy to the recruitment of

termination factors in bacteria, the nascent RNA could work as a loading platform for DNA repair factors. However, the insertion of a selfcleaving ribozyme in a transcribed gene did not impact its repair rates by TC-NER [78], indicating that an intact nascent mRNA is not required for TC-NER repair. Finally, since CSB/Rad26 functions as an elongation factor for both RNAPII and RNAPI transcribed genes, it might travel along with transcribing polymerase and undergo conformational changes upon stalling of the elongating RNAP [64]. These conformational changes, in addition to increasing CSB/Rad26 binding to chromatin, might contribute to signaling the DNA damage and to recruit NER proteins. Further studies should hopefully enlighten our understanding of the mechanisms by which NER factors are efficiently recruited to RNAP stalled at a bulky DNA lesion. It appears that TC-NER functions to ensure a rapid repair of the DNA that is actively needed by the cell. Indeed, several mechanisms exist to resolve the blockage that an elongating polymerase stalled at a DNA lesion represents for gene expression, including backtracking on the DNA template, bypassing the DNA damage or being targeted to proteosomal degradation. Each of these possibilities can be accompanied - or not- by the concomitant action of the NER machinery to remove the DNA lesion. In yeast, UV irradiation stabilizes existing mRNAs [140] and this stabilization might represent an additional mechanism to allow the cell to use the mRNA that were produced before the occurrence of broad DNA damage to full capacity. One of the challenges for the future is to dissect and understand how the decision between these different possibilities is achieved and how the mechanisms are prioritized. Possibly, this decision might be influenced by the specific transcriptional context in each particular case (chromatin structure, associated elongation factors, protein modifications, nature of the DNA lesion, etc.), and the availability of specific factors. Acknowledgments We would like to thank R.E. Wellinger for critical reading of the manuscript and D. Haun for style supervision. Research in AA's laboratory is

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