UVSSA and USP7: new players regulating transcription-coupled nucleotide excision repair in human cells

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes DNA damage located in actively transcribed genes. Defects in TC-NER are associated with several human disorders, including Cockayne syndrome (CS) and ultraviolet (UV)-sensitive syndrome (UVSS). Using exome sequencing, and genetic and proteomic approaches, three recent studies have identified mutations in the UVSSA gene as being responsible for UVSS-A. These findings suggest a new mechanistic model involving UV-stimulated scaffold protein A (UVSSA) and the ubiquitin-specific protease 7 (USP7) in the fate of stalled RNA polymerase II during TC-NER, and provide insights into the diverse clinical features of CS and UVSS.


Genetic characterization of the third complementation group of UV S S patients
Th ree papers published recently in Nature Genetics have reported the isolation of the gene responsible for the UV S S-A complementation group, UVSSA (encoding UVstimulated scaff old protein A), using three diff erent approaches: exome sequencing of cell lines derived from patients with UV S S-A [7], complementation of the repair defect of UV S S-A cells [8] and stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic isolation of diff erentially ubiquitinated proteins following UV irradiation [9].

Abstract
Transcription-coupled nucleotide excision repair (TC-NER) specifi cally removes DNA damage located in actively transcribed genes. Defects in TC-NER are associated with several human disorders, including Cockayne syndrome (CS) and ultraviolet (UV)-sensitive syndrome (UV S S). Using exome sequencing, and genetic and proteomic approaches, three recent studies have identifi ed mutations in the UVSSA gene as being responsible for UV S S-A. These fi ndings suggest a new mechanistic model involving UV-stimulated scaff old protein A (UVSSA) and the ubiquitin-specifi c protease 7 (USP7) in the fate of stalled RNA polymerase II during TC-NER, and provide insights into the diverse clinical features of CS and UV S S.
Keywords DNA repair-defi cient diseases, transcriptioncoupled repair, stalled RNA polymerase, UV sensitivity Using cell lines derived from two UV S S-A patients, Nakazawa et al. [7] identifi ed homozygous mutations (c.367A>T) leading to a premature stop codon in the KIAA1530 (renamed UVSSA) gene. Interestingly, a homo zygous missense mutation (p.Cys32Arg) in this gene was found in an individual previously mis-diagnosed with mild XP. Zhang et al. [8] carried out microcellmediated chromosome transfer of mouse DNA in order to complement the repair defi ciency of UV S S-A human cells, and isolated the mouse homologue of KIAA1530 as the gene responsible for UV S S-A. Sequencing of this gene from several UV S S-A cells also revealed mutations leading to premature termination of the UVSSA protein [8]. Schwertman et al. [9] identifi ed several diff erentially ubiquitinated proteins following UV irradiation of HeLa cells. Th e most prominent factors were repair proteins involved in NER (XPC, DDB2, RNA RNA polymerase (RNA Pol) II and CSB) as well as UVSSA. Using mass spectrometry, it was demonstrated that UVSSA interacts with the deubiquitinating enzyme ubiquitin carboxylterminal hydrolase 7 (also known as ubiquitin-specifi c protease 7, USP7) [8,9]. Transfection of wild-type tagged UVSSA cDNA restored normal RRS in UV S S-A cells, while small interfering RNA (siRNA)-based depletion of UVSSA transcripts caused a marked reduction of RRS in normal cells, illustrating that the UVSSA-USP7 complex is crucial for restoration of gene expression following UV irradiation. Taken together these data indicate that UVSSA is the causal gene in UV S S-A, and that the UVSSA-USP7 complex is involved in TC-NER.

UVSSA interactions and the role of the ubiquitin proteasome pathway
Using three-dimensional structure prediction of UVSSA, Nakazawa et al. [7] identifi ed two domains of unknown function: a VHS domain (homology with the Vps-27, Hrs and STAM domain) near the amino terminus and a DUF2043 domain (EMBL-EBI IPR018610) near the carboxyl terminus. Th e VHS domain has been implicated in ubiquitin binding and in interaction with the carboxyterminal part of RNA Pol II; it has also been suggested that the UV S S-A mutation Cys32Arg might obstruct interactions between the VHS domain and ubiquitinated proteins. UVSSA truncated mutants lacking either VHS or DUF2043 domains failed to complement the UV S S-A defi ciency, indicating that these two domains are necessary for TC-NER activity.
One of the major players in TC-NER is the ten-protein complex TFIIH, involved in both NER and transcription initiation [1]. Nakazawa et al. [7] demonstrated that UVSSA interacts with ERCC2, ERCC3, p62 and the CAK subcomplex (all part of TFIIH). Furthermore, Zhang et al. [8] showed that UVSSA interacts with CSA in the absence of UV, and with CSB and RNA Pol II after UV irradiation, and that both UVSSA and CSB are necessary for full completion of TC-NER. Th us, interaction between UVSSA and the major proteins involved in TC-NER suggests that UVSSA may play a pivotal role in this process.
USP7 has several NER and DNA damage response (DDR) proteins as substrates. Schwertman et al. [9] showed that USP7 resided in chromatin-immunoprecipitated TC-NER complexes in a UV-and UVSSAdependent manner. In UV S S-A cells, the absence of UVSSA correlated with the instability of CSB, probably due to the lack of USP7 recruitment in the TC-NER complex. Indeed, depletion of USP7 by siRNA caused a similar RRS defi ciency and decreased levels of CSB. Th ese data indicate that UVSSA and USP7 cooperate to protect CSB from UV-induced degradation in TC-NER via the ubiquitin-proteasome pathway. Because USP7 has multiple roles in the DDR, an important role of UVSSA might also be to deliver the deubiquitinating enzyme to the vicinity of TC-NER factors, allowing smooth regu lation of ubiquitination and deubiquitination. Zhang et al. [8] also showed that this recruitment is CSA-dependent.
Using local UV damage, it has also been shown that tagged-UVSSA accumulated in vivo at UV-induced DNA lesions (with kinetics similar to CSB), and interacted with the elongating form of RNA Pol II (Pol IIo) [9]. Following UV irradiation, transcription is rapidly inhibited and fast repair of lesions is necessary to ensure survival of damaged cells. Pol IIo is stalled at UV-induced DNA lesions and needs fi rst to be displaced by backtracking or degradation to allow access to repair factors. During this step, Pol IIo is ubiquitinated, and CSA and CSB proteins are necessary for this process. In UV S S-A cells, the ubiquitinated Pol IIo was almost undetectable and the normal Pol IIo form disappeared over a 6-h period following UV irradiation. During the same period, CSB protein was degraded in a proteasome-and UVdependent manner, indicating that UVSSA contributes to the stabilization of the CSB complex during TC-NER [7,9]. Th e absence of UVSSA or USP7, or mutations in the VHS domain, destabilizes Pol IIo [8]. Th ese anomalies were corrected by wild-type UVSSA cDNA transfection, but not with mutants in the VHS domain. During TC-NER, Pol IIo can be dephosphorylated and recycled to Pol IIa, ready to start another round of transcription. In UV S S-A cells, dephosphorylation of Pol IIo is inhibited, as previously found in CS cells [8], and the absence of dephosphorylation does not allow the recycling of Pol II for transcription initiation [7]. Taken together, these fi ndings suggest a new model for UV-induced TC-NER in which UVSSA and USP7 are crucial for Pol IIo ubiquitination and the resumption of normal transcription.

A new model for UV-induced TC-NER
Patients with UV S S exhibit photosensitivity and mild skin abnormalities; in contrast, patients with CS show major growth retardation, abnormal mental development and neurological anomalies, usually leading to early death [3,10] (Table 1). Although both classes of patients are defi cient in UV-induced TC-NER, we have previously shown that CS cells are defi cient in repair of oxidative DNA lesions [6]. In a patient with CSA-mutated UV S S1VI, a missense mutation found in the carboxy-terminal part of the gene, suggested that this mutation does not allow UV-induced TC-NER, but does allow repair of reactive oxygen species (ROS)-induced damage, explaining the mild symptoms [6]. In the new model where the UVSSA and USP7 proteins are taken into account, ubiquitination of stalled RNA Pol IIo at DNA lesions is a necessary step to allow repair [1]. Th e model proposed by Nakazawa et al. [7] suggests that UVSSA recruits an E3 ubiquitin ligase allowing effi cient ubiquitination of Pol IIo. In UV S S-A cells, stalled RNA Pol IIo can still be ubiquitinated by a CSB-and CSAdependent pathway, but cannot be deubiquitinated due to the absence of the UVSSA-USP7 complex [8], so that transcription resumption does not occur and the cells are RRS defi cient. In CS cells (mutated in CSA or CSB) neither of these two processes occurs and stalled Pol IIo is arrested for a longer time, leading to apoptosis and to more severe clinical features as described in Table 1. However, no explanation has been proposed for the paradoxical group of patients with CSB-mutated UV S S [4,5], where the complete absence of the CSB protein gave rise to a mild UV S S phenotype [10]. Th e defective processing of oxidative DNA damage characteristic of CS patients does not occur in patients with CSB-mutated UV S S for reasons still not explained by this new model of TC-NER.
In conclusion, these results suggest a new model for TC-NER involving UVSSA and USP7, and provide new insights into the mechanisms underlying the diff erences in severity of CS and UV S S. UVSSA and USP7 are identifi ed as two new key factors controlling the fate of stalled RNA Pol II, the steady-state level of CSB, the effi ciency of TC-NER and cell survival following DNA damage. Th e data reported indicate that diff erences between severe CS syndromes and mild UV S S are due to diff erences in transcription and/or repair of oxidative DNA damage in transcribed strands. Further investigation of the repair of oxidative damage at the transcriptional level should help us to understand how this process might be involved in progressive neurological deterioration associated with ageing in CS.

Competing interests
The author declares that he has no competing interests.