Transcription factor IIH: A key player in the cellular response to DNA damage

Biochimie 81 (1999) 27−38 © Société française de biochimie et biologie moléculaire / Elsevier, Paris

Transcription factor IIH: A key player in the cellular response to DNA damage Philippe Frit, Etienne Bergmann, Jean-Marc Egly* Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP 163, Université Louis-Pasteur, Strasbourg, 67404 Illkirch cedex, France (Received 21 June 1998; accepted 30 November 1998) Abstract — TFIIH (transcription factor IIH) is a multiprotein complex consisting of nine subunits initially characterized as a basal transcription factor required for initiation of protein-coding RNA synthesis. TFIIH was the first transcription factor shown to harbor several enzymatic activities, likely indicative of functional complexity. This intricacy was further emphasized with the cloning of the genes encoding the different subunits which disclosed direct connections between transcription, DNA repair and cell cycle regulation. In this review, we emphasize those functions of TFIIH involved in DNA repair, as well as their relationship to TFIIH’s roles in transcription, cell cycle control and apoptosis. These connections may prove to be essential for the cellular response to DNA damage. © Société française de biochimie et biologie moléculaire / Elsevier, Paris TFIIH / DNA repair / transcription / cell cycle / apoptosis

1. Introduction Transcription factor IIH (TFIIH), as well as rat (factor δ) and yeast (factor b) homologues were initially identified as class II general transcription factors (reviewed in [1]) absolutely required for in vitro reconstituted transcription system driven by RNA polymerase II (RNA pol II) [2–5]. The function of TFIIH in transcription relies on its multiple enzymatic activities. Indeed, TFIIH was the first general transcription factor found to possess several catalytic activities such as a DNA-dependent ATPase activity linked to a DNA helicase and protein kinase activities. TFIIH is a multiprotein complex consisting of nine subunits ranging from 89 kDa to 32 kDa (table I), which are part of two main subcomplexes, the core-TFIIH composed of five subunits (p89-XPB, p62, p52, p44 and p34), and a ternary subcomplex (cdk7, cyclin H and MAT1) harboring the kinase activity [6–9]. The remaining 80-kDa subunit (XPD) can be found either associated with the core or with the kinase complex [6–9]. The nine genes encoding the different subunits have been cloned, revealing highly conserved structure and function from yeast to * Correspondence and reprints Abbreviations: cdk, cyclin-dependent kinase; CS, Cockayne’s syndrome; CTD, carboxy-terminal domain of the largest RNA pol II subunit; ERCC, excision repair cross-complementing; GGR, global genome repair; MAT1, ménage-à-trois-1; NER, nucleotide excision repair; RNA pol II, RNA polymerase II; TBP, TATA binding protein; TCR, transcription-coupled repair; TFIIH, transcription factor IIH; TTD, trichothiodystrophy; XP, xeroderma pigmentosum.

human [10] (table I). However, some differences exist, concerning for example the kinase subcomplex which only consists of two proteins in Saccharomyces cerevisiae, Kin28 and Ccl1, the homologues of cdk7 and cyclin H, respectively [10–13]. In the course of the systematic cloning of the genes encoding the TFIIH subunits, the most striking result came from the identification of the 89-kDa subunit as XPB [14–16], a protein already known for its involvement in the DNA repair pathway termed nucleotide excision repair (NER). The role for TFIIH in NER was further emphasized after having identified p80 as XPD, another repair protein [16–19]. In addition to the presence of DNA repair proteins within TFIIH, the kinase subcomplex has been demonstrated to correspond to the cdk activating kinase (CAK) which participates in the activation of cyclin-dependent kinases (cdk) required for progression through the cell cycle [20–23]. This discovery disclosed an additional link between TFIIH and cell cycle regulation. Moreover, cells mutated in either the XPB or the XPD gene were deficient in p53-dependent apoptosis, also suggesting an implication of TFIIH in programmed cell death [24]. All these data strengthen the idea that TFIIH may be a key component in the cellular response to DNA damage.

2. TFIIH, a transcription factor The transcription reaction proceeds through different steps: preinitiation, initiation, promoter clearance, elongation and termination. The first step consists of the assembly of the different basal transcription factors (TFIID, IIA,

28

Frit et al.

CAK

Activities

Functions

XPB (Rad25/Ssl2)

89

ATP binding site, helicase motifs, NLS, HTH

DNA dependent ATPase helicase 3'→5'DNA

p53-dependent apoptosis

p62 (Tfb1) p52 (Tfb2) p44t + p44c (Ssll)

62 52 44

NLS Zn finger

DNA binding

XPD regulatory subunit

p34 (Tfb4) XPD (Rad3)

34 80

Zn finger ATP binding site, helicase motifs

p53-dependent apoptosis

Cdk7 (Kin28)

40

ATP binding site, catalytic domain, NLS, T loop

DNA-dependent ATPase, 5'→3'DNA helicase Protein kinase (Cdk1,2,4,6, TBP, CTD, TFIIEα, TFIlFα, RARα)

Cyclin H (Ccl1)

34

Cyclin box

MAT1 (Tfb3/Rig2)

32

RING finger, coiled-coil

CAK catalytic subunit, CTD kinase

Cdk7 regulatory subunit Cdk7 regulatory subunit

Transcription

MW Motifs (kDa)

Cell cycle regulation

Subunits (yeast homologues)

NER

Core

Table I. TFIIH subunits.

NLS, nuclear localization sequence; HTH, helix-turn-helix motif; p44t and p44c differ by three amino acids and correspond to p44 encoded by the telomeric and the centromeric copy of the p44 gene, respectively; RARα, retinoic acid receptor α.

IIB, IIF, IIE and IIH) along with a non-phosphorylated RNA pol II molecule (RNA pol IIA) at the promoter to form the preinitiation complex. An alternative to a sequential assembly model, a one-step loading of a preassembled holoenzyme, has also been proposed [25–27]. This holoenzyme, carrying a battery of factors required not only for transcription but also for DNA repair [26], will join the promoter already bound by TFIID and TFIIB to form the inactive closed preinitiation complex. ATP hydrolysis will then promote the opening of DNA to allow the formation of the first phosphodiester bond and the release of RNA pol II from the promoter to carry on with transcript elongation. The intermediate step (between initiation and elongation), during which RNA pol II is phosphorylated on the carboxy-terminal domain (CTD) of its largest subunit and disengages from the initiation complex, is referred to as promoter clearance. Based on the ATPase/DNA helicase activity of TFIIH, and given the facts that a supercoiled DNA matrix [28, 29] or an artificially pre-melted promoter [29–32] circumvents the requirement of ATP hydrolysis and the presence of TFIIH for transcription initiation, it was suggested that the primary role of TFIIH in (pre)initiation probably consisted of promoter opening over the start site. This would then allow access of RNA pol II to the template strand for the formation of the first phosphodiester bond [33, 34]. The various events associated with the first phosphodiester bond formation and promoter clearance require unwinding activity and ATP as a source of energy [35–37].

Among the basal transcription factors, only TFIIH is able to fulfil such a role. Indeed, it possesses two subunits, XPB and XPD (table I), which harbor ATP-dependent DNA helicase activities. Moreover, evidence from genetic and biochemical studies in yeast indicates that one of them, the XPB helicase activity, is essential for transcription [16, 38, 39], whereas in humans, mutations in both helicases can lead to transcription syndrome phenotypes (see below). Alternatively (or in addition), the possibility for the ATP requirement during the transition from initiation to elongation to be associated with the phosphorylation process of the carboxy-terminal domain (CTD) of the largest subunit of RNA pol II, remains an interesting point. Indeed, TFIIH, which possesses a cyclin-dependent kinase activity (cdk7), has been shown to be responsible for the CTD phosphorylation [11, 22, 23, 40–42] which was suggested to occur immediately prior to, or concomitantly with promoter clearance [43] (for a review on CTD phosphorylation, see [44]). The significance of CTD phosphorylation is not well established and several hypotheses have been proposed in addition to promoting the release of RNA pol II from the transcription initiation factors bound to the promoter [45]. Indeed, it has to be pointed out that the hyperphosphorylated form of RNA pol II (RNA pol IIO) is not exclusively associated with transcription elongation since it is also found in non-chromosomal structures [46]. RNA pol IIO seems instead to be associated

TFIIH and cellular response to DNA damage with a function in mRNA processing, as judged by specific interactions between the phosphorylated CTD and 5'capping enzymes [47–49], splicing factors [50–53] and cleavage-polyadenylation factors [54]. Moreover, disrupting these interactions in vitro with anti-CTD antibodies [50], or in vivo by truncating the CTD [47, 54] and overexpressing CTD peptide competitors [51] results in the inhibition of pre-mRNA 5'-capping, splicing and 3'-processing reactions. In the light of these observations, it cannot be ruled out that the function of RNA pol II in mRNA processing might affect indirectly the overall RNA synthesis rate. The role of TFIIH in transcript elongation following the promoter clearance step is more controversial. On the one hand, it was demonstrated that RNA pol II can support RNA extension without TFIIH [35, 55]. On the other hand, transcription elongation was found to be inhibited by DRB, a kinase inhibitor [56], or anti-TFIIH antibodies [57]. This might reflect the requirement for RNA pol II to remain in a hyperphosphorylated form (RNA pol IIO) to achieve elongation. As a result, CTD phosphatase [58] and CTD kinase activities could act as additional regulatory components for the transcription reaction. Finally, transient interactions between TFIIH and elongating RNA pol II molecules could facilitate the function of TFIIH in the monitoring of genome integrity, and particularly in preferential repair of actively transcribed genes (see below). 3. TFIIH and DNA repair 3. 1. TFIIH in nucleotide excision repair (NER) To remove DNA lesions in response to genotoxic injuries interfering with cellular functions such as transcription and replication, several DNA repair processes have been evolved. They proceed essentially through the direct reversion of the lesion or through the excision of the damage and the subsequent replacement by a newly synthesized intact DNA (for a detailed review on the different DNA repair processes, see [59]). Among the various DNA repair pathways, nucleotide excision repair (NER) plays an essential role in cell survival by removing a wide range of DNA lesions exemplified by the major UV-induced photoproducts. NER proceeds through different steps highly conserved during evolution from bacteria to human: damage recognition, incision on each side of the lesion, excision of a 30-mer oligonucleotide bearing the lesion, resynthesis and ligation (for review, see [60]). In man, our understanding of NER was facilitated by the availability not only of repair-deficient rodent cell lines established by induced mutagenesis, but also of cells derived from patients suffering from recessive hereditary repair syndromes (reviewed in [61]). These disorders are typified by the xeroderma pigmentosum (XP) which is

29 characterized by sunlight hypersensitivity and a greatly increased incidence of UV-induced skin cancers, further emphasizing the importance of such a DNA repair pathway in genetic integrity. Following complementation analysis by cell fusion, XP cells have been shown to fall into seven genetic complementation groups (XP-A to XP-G), two of them, XP-B and XP-D, corresponding to cells mutated in genes encoding TFIIH subunits. The NER reaction is now well characterized and can be reconstituted in vitro with recombinant or purified factors including XPA-RPA (a DNA damage recognition complex composed of XPA and replication protein A), XPCHHR23B (a DNA damage recognition complex composed of XPC and a human homologue of Rad23), TFIIH, XPG (a structure-specific nuclease cutting on the 3' side of the lesion), XPF-ERCC1 (a nuclease complex composed of XPF and excision repair cross-complementing-1 protein and cutting on the 5' (side of the lesion), RFC (replication factor C), PCNA (proliferating cell nuclear antigen), DNA polymerase δ/e and DNA ligase I [62–64]. Such in vitro studies revealed that XPB and XPD (as well as their counterparts in yeast) participate in the NER process as part of the TFIIH multiprotein complex. Consistently, TFIIH is able to complement in vivo and in vitro the repair deficiencies of XP-B and XP-D cells or cell extracts [18, 19, 65], whereas the purified Rad3 protein, the yeast homologue of XPD, does not complement in vitro the repair activity of protein extracts from rad3 mutant cells [66]. However, in the same conditions, the Ssl2 protein, the homologue of XPB, can partially correct the repair deficiency of ssl2 mutant protein extracts, probably reflecting its capacity to substitute to some extent for the mutated form of Ssl2 in TFIIH [66]. Ssl2 is indeed less tightly bound than Rad3 to TFIIH [11, 16] which contrasts with the symmetrical situation in human in which XPD can readily be dissociated from TFIIH and complement the repair defect of XP-D extracts as part of a complex with CAK [8]. These observations raise the question of the role of each TFIIH subunit in NER. According to the UVsensitivity phenotype of the different mutant cells in yeast and human, at least five TFIIH subunits could be considered as repair proteins: the XPB/Rad25 and XPD/Rad3 helicases, as well as p62/Tfb1 [67, 68], p44/Ssl1 [68, 69] and p52/Tfb2 [10]. It has to be noted that all of these subunits are components of the core-TFIIH/XPD complex. Expectedly, immunodepletion or experiments with anti-p34 antibodies or anti-p52 antibodies led to the inhibition of NER activity in vitro and in vivo [70, 71]. However, with respect to identifying repair factors, inhibition experiments with antibodies also reveal indirect effects on core-TFIIH activity since anti-cdk7 antibodies gave similar results [21], although the kinase complex was shown to be dispensable for NER activity in human [72] and yeast [13, 73]. Consistent with the latter observations, yeast kin28 (the yeast homologue of cdk7) and rig2 (the

30 yeast homologue of MAT1) mutant strains only exhibit a mild, if any, UV-sensitivity [42, 74]. Consequently, only XPD and the subunits belonging to the core-TFIIH are believed to participate in NER [75], either in a quite direct manner for the subunits harboring enzymatic activities (XPB and XPD), or probably more indirectly as regulatory or assembly factors for the remaining less characterized subunits. For instance, p44 has recently been found to exert a stimulatory effect on the XPD helicase activity [76]. The mutations found in XPB and XPD genes could affect differently the helicase activities and/or the overall conformation of TFIIH which could be detrimental for the function(s) of TFIIH. Having established the in vitro and in vivo requirement of TFIIH for NER, the question arises of how TFIIH participates in the repair reaction. TFIIH has been proved to participate in the NER reaction through its enzymatic activities and protein-protein interactions with other repair factors. The helicase activities of XPB [39, 73, 77] and XPD [73, 78] are both required for NER as they allow the opening of DNA around the lesion. This open structure seems to be essential for incision on the 3' side by XPG and on the 5' side by the XPF-ERCC1 complex [79, 80]. In addition to be required for the incision step, the DNA helicase activity of Rad3 in yeast was found to be inhibited by DNA lesions [81], and Rad3 by its own was shown to bind specifically to (6–4) photoproducts depending on DNA superhelicity [82], which suggests a role for TFIIH at the damage recognition step. Apart from its enzymatic activities and according to the pre-assembled repairosome model [13] or to the sequential assembly model of a repair complex at a damage site [83], additional implications of TFIIH in the NER process are likely to rely on its interactions with other repair factors. For instance, TFIIH can interact with the DNA damage recognition factor XPA [84]. This interaction becomes stronger when XPA is already bound to UV-induced DNA lesions [85]. TFIIH was also shown to interact with XPC (or Rad4 in yeast) [19, 86]. XPC binds specifically damaged DNA [87] and single-stranded DNA, and is essential for DNA unwinding by TFIIH [80], probably by promoting and/or stabilizing the open complex around the lesion [88, 89]. In addition to opening the double helix which enables the double incision around the lesion, TFIIH may act more directly in the incision step by recruiting the endonuclease XPG (or Rad2 in yeast) [86, 90, 91] or by stimulating the 5'-incision activity of XPF-ERCC1 [80]. 3.2. Transcription-coupled repair NER activity is heterogeneous throughout the genome as it proceeds more rapidly on transcribed genes than in inactive chromatin [92] (reviewed in [93]). This phenomenon referred to as preferential repair or transcriptioncoupled repair (TCR) (by opposition to the so-called

Frit et al. global genome repair or GGR), relies mainly on a more rapid damage removal on transcribed strands of active genes than on non-transcribed strands [94]. This phenomenon functionally links transcription to NER and therefore makes TFIIH a compelling candidate for an essential role in TCR. The TCR mechanism is conserved from bacteria to human. In Escherichia coli, a single transcription-repair coupling factor (TRCF) encoded by the mfd gene has been isolated [95]. In vitro experiments demonstrated that TRCF recognizes an RNA polymerase arrested at a DNA lesion on the transcribed strand, removes this stalled RNA polymerase, and recruits the NER machinery at the damage site, thus facilitating the repair reaction [96]. In humans, the mechanism underlying this coupling seems to be much more complex. A function similar to that fulfilled by TRCF is probably carried out, at least partly, by CSA and CSB proteins whose corresponding genes, when mutated, give rise to the Cockayne syndrome (CS) [61]. The repair defect of CS cells specifically affects the TCR, i.e., CS cells do not repair transcribed genes at higher rate and exhibit no strand bias between the transcribed and the non-transcribed strands, but perform GGR at a normal level [97]. In contrast to XP, the Cockayne syndrome is not a cancer-prone hereditary disease, but is characterized by developmental retardation and neurological abnormalities. Such clinical features cannot be simply accounted for by a DNA repair defect but are better understood considering CS as a transcription syndrome [98] (see also section 3.3.). Consistent with this hypothesis are the findings that transcriptional activity is reduced in CS cells in vivo [99] and in vitro [100], and that CSB interacts with RNA pol II [101, 102] and stimulates transcription elongation [103]. CS cells fall into five complementation groups and despite a clinical heterogeneity between XP and CS, a striking genetic overlapping underlies these diseases. Indeed, the CS phenotype also results from mutations in XPB, XPD and XPG genes which in fact lead to mixed syndromes combining both XP and CS clinical features. The finding that mutations in two subunits of TFIIH can lead to CS features further emphasizes the implication of TFIIH in TCR. This may reflect the importance of interactions between TFIIH, CSA and CSB, and putatively, the existence of a ‘TCR complex’ containing these factors [90, 104]. As mentioned above, TCR is believed to proceed in eukaryotes in a similar manner as in bacteria, via an activity recognizing the stalled RNA pol II and then recruiting the NER machinery. CSB could be considered as the human counterpart of TRCF since both factors possess ATPase activity [105, 106, 107] and helicase motifs [96, 108, 109]. Since TFIIH is not associated with RNA pol II during elongation [55], it has to be recruited together with other repair factors to the arrested RNA pol II. CSB may participate to this recruitment as part of the

TFIIH and cellular response to DNA damage elongation complex [101, 110]. Consistently, repair of the transcribed strand immediately downstream of the start site, i.e., a region where TFIIH is still associated with RNA pol II, does not depend on CSB (or Rad26, the yeast homologue) [111, 112]. Similar results have been reported recently for the CSA factor [113]. In addition to CS proteins, TCR is believed to involve the same DNA repair machinery as GGR, with the exception of XPC. Indeed, XP-C cells are specifically deficient for GGR but not for TCR [114]. Conceivably, a transcription bubble blocked at a damage site would make dispensable the function of XPC in recognizing the damage and promoting or stabilizing DNA opening by TFIIH around the lesion prior to the dual incision [80, 87, 88]. Nevertheless, many questions on the mechanism of TCR remain unsolved, particularly concerning the fate of RNA pol II stalled at a damage site. In one model, RNA pol II was demonstrated to move back from the lesion and cleave the 3'-terminus of nascent RNA upon activation by the transcription elongation factor SII [115], allowing NER to take place and subsequent resumption of transcript elongation. However, and in contrast to E. coli RNA polymerase, RNA pol II arrested at a DNA lesion does not impede excision repair [116, 117]. Alternatively, in vitro studies with cell extracts revealed that RNA pol II was removed from the damaged template independently of CSA or CSB [117]. Accordingly, CSB alone in a reconstituted system is unable to remove the arrested RNA pol II [107]. These discrepancies and the difficulty in reproducing TCR in vitro with yeast or human factors could reflect the requirement for additional activities in eukaryotes. In this respect, recent studies provide evidence supporting a role for ubiquitination of RNA pol II. Indeed, following cell exposure to cis-platin or UV-radiation, a fraction of the RNA pol II, corresponding to the phosphorylated elongating form, was found to be ubiquitinated, leading to its degradation by the proteasome [118, 119]. Consequently, and in contrast to the backward/resumption model, transcription elongation would abort rather than resume after removal of the lesion. The ubiquitination of RNA pol II depends on CSA and CSB. One possibility is that CSdependent ubiquitination of RNA pol II is a consequence of transcriptional arrest, reflecting a role for CS proteins in transcription irrespective of their role in TCR. Alternatively, this could suggest a specific and active role for RNA pol II ubiquitination in TCR, for example by providing a recognition signal for driving repair proteins to DNA damage that blocks transcription. Finally, the links between transcription and ubiquitin-mediated proteolysis are not unprecedented, and the physical interaction between XPB and SUG1, a component of the 26S proteasome [120], further suggests a role for RNA pol II ubiquitination in TCR.

31 3.3. Connections between transcription and DNA repair Considering TFIIH as a multiprotein complex engaged both in transcription and DNA repair, it is conceivable that transcription, NER and TCR could be variously affected, depending on the subunit of TFIIH mutated and on the nature of the mutation. In this regard, as mentioned above, the peculiar clinical features of CS patients are better understood when considering CS as a transcription syndrome. In the same way, trichothiodystrophy (TTD), another non-cancer-prone inherited repair disease characterized by brittle hair and nails associated with deficient sulfur-rich protein synthesis [61], may also be considered as a transcription syndrome [121]. TTD cells fall into three complementation groups. Similarly to what has been described for CS, TTD can result not only from mutations in the yet unidentified TTDA gene, but also from mutations in XPB and XPD genes. Curiously, the repair defect of TTD-A cells was complemented by microinjection of purified TFIIH [121], but none of the TFIIH subunits tested was shown to be mutated in these cells. In some aspects, this is reminiscent of the yeast MMS19 gene whose deletion leads to NER and transcription defects which are complemented in vitro with purified TFIIH but not with purified Mms19 protein [122, 123]. Mms19 has been therefore proposed to participate indirectly in NER and transcription by regulating TFIIH activity. Regarding the genetic links between XP, CS and TTD, it appears surprising that among the nine TFIIH subunits, only XPB and XPD are found mutated in human diseases. This could reflect the essential role for helicase activities in transcription and NER. One possibility is that the remaining subunits do not tolerate mutations. It cannot be ruled out however, that mutations exist, being either silent or associated with other syndromes whose connections with TFIIH have not been established yet. In this regard, Werdnig-Hoffmann disease, the severe form of spinal muscular atrophy (SMA), has been shown to be associated with large-scale deletions on chromosome 5q11.213.3 [124], a region containing the telomeric copy of the duplicated gene encoding p44-TFIIH (p44t) [125, 126]. However, the lack of p44t does not alter TFIIH activity in transcription and NER [125]. The findings that TFIIH as well as the core-TFIIH are both efficient in repair whereas only TFIIH can perform transcription [13, 72, 73], raise the possibility of the existence of multiple forms of TFIIH, each endowed with specific function(s). However, the transition from one form to another would enable competition to occur between transcription and NER [13]. In vitro transcription/ NER systems using human cell-free extracts failed to reveal such a TFIIH-dependent competition [127–129]. In contrast, in a yeast system, NER competed efficiently (but partially) with transcription due to the preferential mobilization of TFIIH for NER [130]. This phenomenon was strictly dependent on Rad26, therefore suggesting a role

32 for Rad26 (and CSB) in the assembly/disassembly of TFIIH or in the down-regulation of transcriptionproficient forms of TFIIH until NER is achieved. These observations could explain, at least in part, why transcription is inhibited when cells undergo DNA damaging treatments. In CS cells, this transcriptional inhibition persists, even in the case of efficiently repaired lesions such as acetylaminofluorene (AAF)-induced DNA adducts which are mainly removed by GGR [131, 132]. These data were interpreted by proposing CS proteins to act as repair-transcription uncoupling factors, i.e., CS proteins would convert back TFIIH from a repair-proficient status to a transcription-active form. Such a model agrees well with the fact that transcription is inhibited even at low lesion density, suggestive of an inhibition of transcription initiation rather than elongation [132]. However, it is noteworthy that TFIIH might not be the unique transcription factor involved in this mechanism of inhibition. Indeed, recent studies have demonstrated the direct interaction between TBP (TATA-binding protein) and certain DNA damage such as UV-, cis-platin- or AAF-induced DNA lesions, resulting in TBP sequestration and inhibition of transcription in vitro and in vivo [128, 129]. In this respect, it cannot be ruled out that a portion of TFIIH could be also sequestered on DNA lesions through protein-protein interactions between transcription factors resulting in the assembly of a ‘transcription complex’. Even though these data make the situation more complicated, they will have to be kept in mind to elucidate the precise role of TFIIH in cell response to DNA damage. 4. TFIIH and cell cycle In addition to its role in transcription and NER, TFIIH has been suspected to be involved in cell cycle regulation as soon as it appeared to contain a cyclin-dependent kinase, cdk7 [11, 21]. Cell cycle progression through the different phases (G1, S, G2 and M) is mediated by a family of serine-threonine kinases called cyclin-dependent kinases (cdk) (reviewed in [133]). In order to be active, cdks require, first, to be associated with a regulatory subunit belonging to the cyclin family, and second, to be phosphorylated on a conserved threonine residue corresponding to threonine 161 in human p34cdc2/cdk1. This phosphorylation event, which confers high kinase activity to the complex and strengthens the interaction between the two partners, can be brought about by CAK (cdk activating kinase), which belongs itself to the cdk family. Cdk7 is the catalytic subunit of CAK, and is not only associated with a cyclin subunit, the cyclin H [20], but also with another protein, MAT1 (‘ménage-à-trois-1’), a member of the RING finger protein family which promotes stable association between cdk7 and cyclin H, and the subsequent activation of cdk7, regardless of its phosphorylation state [6, 134-136]. Both cdk7 partners are also part of the TFIIH complex [6, 22, 23].

Frit et al. XPD, which interacts directly with MAT1 in baculovirus coinfection experiments, is thought to anchor CAK to the core-TFIIH [9]. In agreement with this model, three subcomplexes of TFIIH, in addition to the entire complex, have been isolated using classic chromatographic procedures: free CAK, CAK-XPD and core-TFIIH including XPD [4, 6–8]. Interestingly, the substrate specificity of the kinase differs depending on whether it resides in TFIIH or exists as free CAK [9, 137]. Thus, within TFIIH, CAK is able to phosphorylate the CTD of RNA pol II, TFIIEα (p56) and TFIIFα (RAP74), whereas free CAK seems unable to perform this role. On the contrary, this latter shows a strong preference for the phosphorylation of cdk2. Free CAK seems therefore to be devoted to cell cycle regulation whereas CAK within TFIIH promotes transcription reaction. It has been proposed that regulation of TFIIH function in the cell could depend on an equilibrium state between free CAK (and/or CAK-XPD) and TFIIH [9, 137]. Recent data challenged the identification of cdk7 as the physiological CAK in higher eukaryotes. First, studies in Saccharomyces cerevisiae revealed crucial variations: although Kin28, the homologue of human cdk7, plays a role in transcription regulation and possesses CTD kinase activity in vitro, this protein lacks CAK activity [11, 42, 138]. Second, this latter function has been attributed to a protein active as a monomer, Civ1 (CAK in vivo) or CAK1, by virtue of its ability to tightly bind and phosphorylate Cdc28, the homologue of cdk1 in budding yeast, in vitro and in vivo [139–141]. Another argument spreading doubt on the actual role of cdk7/cyclin H as a CAK complex results from the observation that cdk7 is nuclear at all phases of the cell cycle, with the exception of mitosis, whereas one of the CAK substrates, cdk1/cyclin B, resides in the cytoplasmic compartment. Moreover, CAK activity as well as the levels of all three subunits remain constant throughout the cell cycle [142]. Similar results have been obtained with TFIIH [6], but they contrast with the recent report that TFIIH-associated kinase activity was repressed at mitosis, probably via phosphorylation of p62 and/or a 36 kDa TFIIH subunit by cdk1/cyclin B kinase [143]. Indeed, the concomitant down-regulated transcription could be restored in mitotic extracts by adding back TFIIH. If cdk7 is not the catalytic CAK subunit in higher eukaryotes, one simple explanation could be the existence of a CAK1 homologue, but such a kinase has not been identified to date. Moreover, some recent studies strongly suggest that the mechanism used by metazoans to control the cell cycle could differ from the one in budding yeast, arguing for a bifunctionality of the cdk7 enzyme. First, in Schizosaccharomyces pombe, a yeast generally considered to be closer to metazoans in evolution than budding yeast, Mop1/Crk1, the homologue of cdk7, in association with the cyclin H-related cyclin Mcs2, displays CAK activity in vitro as well as CTD kinase activity [144, 145]. Second, in

TFIIH and cellular response to DNA damage Xenopus, the immunodepletion of cdk7 in cycling egg extracts drastically reduces CAK activity. Cdk1 activation in depleted extracts can be restored by specific translation of mRNAs encoding cdk7 and its associated subunits [146]. Third, consistent results have emerged from studies in Drosophila melanogaster, null and temperaturesensitive cdk7 mutants of which show phenotypes very similar to those of equivalent cdk1 mutants. The reduced level of cdk1 phosphorylation in cdk7ts mutants and the rescue with purified cdk7/cyclin H complexes suggest that cdk7 is essential for in vivo CAK activity. Altogether, these data provide strong evidence supporting that a single kinase, cdk7/cyclin H, can perform different fundamental cell functions in higher eukaryotes [147]. Another link between TFIIH, NER and the cell cycle has been established by discovering that the basal transcription factor could interact physically and functionally with the tumour suppressor factor p53. Generally considered as a safeguard of the genome integrity in mammalian cells, this transcriptional trans-activator plays an important role in cell cycle regulation under conditions of

33 genomic stress (reviewed in [148]). In particular, its accumulation after UV irradiation is associated to cell cycle arrest in the G1 phase, probably due to transcriptional activation of the p21CIP1/WAF1 gene, encoding a cdk inhibitor protein. p53 contacts three different subunits ot the TFIIH factor: its amino-terminal transactivation domain interacts with p62 [149, 150], while its carboxyterminus binds directly to the amino-terminal half of XPD [150] and to helicase motif III of XPB [151]. Consequently, p53 is able to inhibit TFIIH helicase activity even though its ATPase activity remains unaffected. Physiological significance of this alteration still has to be determined. Interestingly, p53 mutants frequently found in tumour cells are less efficient TFIIH helicase inhibitors [150, 151]. Conversely, it has been shown that the cdk7/cyclin H complex was able to phosphorylate p53 in vitro, in a manner that is strongly stimulated by MAT1 which also interacts with p53. This phosphorylation of p53 enhances its specific DNA-binding activity in vitro. A model has thus been proposed in which TFIIH could link transcription pause at a lesion site to the cell cycle arrest

Figure 1. A model for a pivotal role of TFIIH in coordinating the cellular response to DNA damage. Following DNA damage, the functions of TFIIH in transcription and cell cycle control would be reduced or turned off, thus contributing to transcriptional inhibition and cell cycle arrest. Conversely, the function of TFIIH in DNA repair and particularly TCR would be facilitated. Finally, depending on the extent of DNA damage, TFIIH could also participate in apoptosis.

34 via phosphorylation (and activation) of p53 [152, 153]. Interestingly, UV irradiation has been previously reported to reduce kinase activity of TFIIH-associated CAK whereas free CAK activity remains unchanged [6]. The recent finding that the cdk inhibitor p16INK4A could interact with TFIIH and RNA pol II CTD, resulting in the inhibition of CTD phosphorylation by the TFIIH kinase [154], further reinforces the link between TFIIH and the cell cycle regulation. It has been a general notion that, as an alternative response to DNA damage, p53 induces apoptosis in certain types of cells, probably mediating its tumour suppressor property. Whereas apoptosis could be induced in normal human fibroblasts by introduction of a wild-type p53 expression vector, this was not the case in XP-B and XP-D mutant fibroblasts unless the wild-type XPB or XPD gene, respectively, was further transfered into the cells [24]. This result directly implicates TFIIH, via its XPB and XPD helicase subunits, in the p53-mediated apoptosis.

5. Conclusion With the discovery of the multiple functions of TFIIH, this factor has emerged as a pivotal component of the cellular response to DNA damage, and probably plays an essential role in coordinating transcription, DNA repair, cell cycle progression and apoptosis (figure 1). Conceivably, following DNA damage, the cell would reduce its transcriptional activity and would concentrate on repairing the lesions, preferentially those at arrested RNA polymerases. This switch of TFIIH from a function in transcription to a function in (preferential) DNA repair, as well as in cell cycle regulation and apoptosis signalling remains, however, to be firmly established and characterized, for example by identifying the different forms (subunit composition) of TFIIH, their putative posttranslational modifications and their specific partners. Another challenging question will be to precisely determine the intermediate signals linking the DNA damaging treatment and the modulation of TFIIH functions.

Acknowledgments We thank Brendan Bell for his critical reading of the manuscript. We are very grateful to the different students and post-docs of the lab for their contribution to the study of TFIIH. Work on this review was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg and grants from the Ministère de la Recherche et de l’Enseignement Supérieur, Human Frontier Science Program, and the Association pour la Recherche sur le Cancer.

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