Ubiquitination in the UV-induced DNA Damage Response: from proteomics to patient

__Abstract__ The integrity of DNA is continuously challenged by genotoxic agents from both internal and external origin that severely hamper vital DNA-dependent processes as genome duplication by replication and reading of the genetic code by transcription. The adverse effects of DNA damage are counteracted by a complex network of genome defence processes, referred to as the DNA damage response (DDR), which consists of different dedicated DNA repair systems and signalling pathways. Nucleotide excision repair (NER) is the main DNA repair process in mammalian cells that removes UV-induced DNA lesions. Protein ubiquitination has emerged as a key regulatory mechanism for this pathway. However, how the entire UV-light induced DDR (UV-DDR) is controlled via ubiquitination remains largely unknown. The aim of the research described in this thesis is to better understand the ubiquitin-mediated regulation of the UV-DDR. To identify new ubiquitin modifications and proteins not previously known to be involved within the UV-DDR on a proteome-wide scale mass spectrometry (MS) was used. To provide the necessary background Chapter 1 summarizes the current knowledge on DDR, ubiquitination and MS-based methods

FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway

Nucleotide excision repair–induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response

The epigenetic mark indicative of DNA UV damage or double-strand breaks is achieved via a common pathway regardless of the cause of damage

UVSSA and USP7, a new couple in transcription-coupled DNA repair

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes transcription-blocking lesions from our genome. Defects in this pathway are associated with two human disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite a similar cellular defect in the UV DNA damage response, patients with these syndromes exhibit strikingly distinct symptoms; CS patients display severe developmental, neurological, and premature aging features, whereas the phenotype of UVSS patients is mostly restricted to UV hypersensitivity. The exact molecular mechanism behind these clinical differences is still unknown; however, they might be explained by additional functions of CS proteins beyond TC-NER. A short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review. In addition, we will focus on two new players involved in TC-NER which were recently identified: UV-stimulated scaffold protein A (UVSSA) and ubiquitin-specific protease 7 (USP7). UVSSA has been found to be the causative gene for UVSS and, together with USP7, is implicated in regulating TC-NER activity. We will discuss the function of UVSSA and USP7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between UVSS and the more severe CS

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

UV-sensitive syndrome (UVSS) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma1, 2, 3, 4. Despite mild clinical features, cells from individuals with UVSS, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER)2, 4, 5, which removes DNA damage in actively transcribed genes6. Three of the seven known UVSS cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively)7, 8. The remaining four individuals with UVSS, one of whom is described for the first time here, formed a separate UVSS-A complementation group1, 9, 10; however, the responsible gene was unknown. Using exome sequencing11, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UVSS-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes

AbstractProtein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein–protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states

UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair

Transcription-coupled nucleotide-excision repair (TC-NER) is a subpathway of NER that efficiently removes the highly toxic RNA polymerase II blocking lesions in DNA. Defective TC-NER gives rise to the human disorders Cockayne syndrome and UV-sensitive syndrome (UV S S) . NER initiating factors are known to be regulated by ubiquitination 2 . Using a SILACbased proteomic approach, we identified UVSSA (formerly known as KIAA530) as part of a UV-induced ubiquitinated protein complex. Knockdown of UVSSA resulted in TC-NER deficiency. UVSSA was found to be the causative gene for UV S S, an unresolved NER deficiency disorder 3 . The UVSSA protein interacts with elongating RNA polymerase II, localizes specifically to UV-induced lesions, resides in chromatinassociated TC-NER complexes and is implicated in stabilizing the TC-NER master organizing protein ERCC6 (also known as CSB) by delivering the deubiquitinating enzyme USP7 to TC-NER complexes. Together, these findings indicate that UVSSA-USP7-mediated stabilization of ERCC6 represents a critical regulatory mechanism of TC-NER in restoring gene expression. Nucleotide-excision repair removes a wide range of DNA damage, including UV-induced lesions. Inherited NER defects lead to extreme cancer proneness (xeroderma pigmentosum) or dramatic premature aging (Cockayne syndrome), showing the clinical impact of NER 4 . NER is initiated by two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER). DNA helix-distorting injuries located throughout the genome are repaired by GG-NER to avoid replication-induced mutations and resultant cancer. TC-NER targets transcription-blocking lesions to enable recovery of arrested transcription, thereby preventing damageinduced apoptosis and resultant aging. In addition, it was shown that TC-NER is also important in overcoming UV-induced transcriptionassociated mutations NER is regulated in response to UV irradiation by ubiquitination 2 of the process-initiating factors xeroderma pigmentosum group C (XPC) 2 ADVANCE ONLINE PUBLICATION Nature GeNetics l e t t e r s TC-NER-deficient cells To investigate the in vivo role of UVSSA, we measured the dynamic association of UVSSA with NER in living cells. Green fluorescent protein (GFP)-tagged UVSSA, which was shown to be biologically active ( Nature GeNetics ADVANCE ONLINE PUBLICATION 3 l e t t e r s with . In contrast, α-amanitin released RNA Pol II from DNA, which also resulted in increased mobility of UVSSA. The opposing effects by these transcription inhibitors on GFP-UVSSA mobility in living cells cannot be caused by an indirect UVSSA interaction with RNA Pol II via ERCC6 (ref. 25), as similar effects were found in CS-B cells To examine whether UVSSA is also present in active, chromatinbound TC-NER complexes via its interaction with RNA Pol IIo, we performed ChIP experiments with antibodies against HGMN1, a chromatin remodeler that is enriched in lesion-stalled TC-NER complexes ADVANCE ONLINE PUBLICATION Nature GeNetics l e t t e r s Whether this RNA Pol II recovery is a direct effect of UVSSA or an indirect consequence of rescued TC-NER remains to be determined. In order to examine how UVSSA influences ERCC6 protein stability, we immunoprecipitated GFP-UVSSA and analyzed UVSSAinteracting proteins by mass spectrometry. Of note, we identified the deubiquitinating enzyme ubiquitin carboxyl-terminal hydrolase 7 (USP7, also called HAUSP) that is known to have various functions in DDR In summary, within our mass spectrometry analysis of the UVinduced ubiquitinome, we identified an uncharacterized protein (UVSSA), which was highly enriched upon UV irradiation in immunopurified ubiquitinated protein complexes. Functional analysis indicated that UVSSA is a new factor implicated in TC-NER and, of note, is the causative gene in the unresolved UV S S-A NER disorder. Two accompanying studies aimed at finding the genetic defect in UV S S-A also identified UVSSA as the gene mutated in individuals with UV S S-A 20,21 . Their further functional analysis is consistent with our observation that UVSSA has an important role in TC-NER and is part of the protein complex containing the elongating form of RNA Pol II. Our data argue for a UV-independent UVSSA-RNA Pol IIo interaction. In contrast to GFP-UVSSA, we were not able to observe RNA Pol IIo accumulation at LUD with current technology. The transient or low-affinity interaction between UVSSA and RNA Pol IIo might be stabilized upon UV irradiation, explaining the UV dependency observed in native immunoprecipitations 20 and the UV independence of the fixed interactions by cross-linking in ChIP shown here. We propose a model Nature GeNetics ADVANCE ONLINE PUBLICATION 5 l e t t e r s Pol II after UV damage 21 seems to imply that UVSSA has functions in TC-NER beyond being a specific shuttle protein for USP7. The causative genes for the two TC-NER defective disorders Cockayne syndrome and UV S S (ERCC6, ERCC8 and UVSSA) are all cofactors of lesion-stalled RNA Pol II. However, the phenotypes of these two TC-NER deficiencies are strikingly different; Cockayne syndrome is characterized by severe neurological and developmental abnormalities in conjunction with UV sensitivity, whereas individuals with UV S S mainly exhibit sun sensitivity, without any clear additional complications METhoDS Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/. Note: Supplementary information is available on the Nature Genetics website. ACknoWLeDgMentS We thank R. Bernards and M. Epping (Nederlands Kanker Instituut) for the Myc-tagged USP7 expression construct and P. Verrijzer and A. Reddy (Erasmus Medical Centre) for shUSP7-expressing lentivirus. We thank H. Slor (Tel Aviv University) for the TA-24sv40 cell line and N.G.J. Jaspers and H. Lans for discussions and critical reading of the manuscript. This work was funded by the Netherlands Genomics Initiative NPCII (to P.S.), 935.19.021 and 935.11.042 (to W.V., C.L. and J.A.M.), the Dutch Organization for Scientific Research ZonMW Veni Grant (917.96.120 to J.A.M.) and TOP grant (912.08.031 to W.V.), Marie Curi

UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair

Transcription-coupled nucleotide-excision repair (TC-NER) is a subpathway of NER that efficiently removes the highly toxic RNA polymerase II blocking lesions in DNA. Defective TC-NER gives rise to the human disorders Cockayne syndrome and UV-sensitive syndrome (UVSS)(1). NER initiating factors are known to be regulated by ubiquitination(2). Using a SILAC-based proteomic approach, we identified UVSSA (formerly known as KIAA1530) as part of a UV-induced ubiquitinated protein complex. Knockdown of UVSSA resulted in TC-NER deficiency. UVSSA was found to be the causative gene for UVSS, an unresolved NER deficiency disorder(3). The UVSSA protein interacts with elongating RNA polymerase II, localizes specifically to UV-induced lesions, resides in chromatin-associated TC-NER complexes and is implicated in stabilizing the TC-NER master organizing protein ERCC6 (also known as CSB) by delivering the deubiquitinating enzyme USP7 to TC-NER complexes. Together, these findings indicate that UVSSA-USP7-mediated stabilization of ERCC6 represents a critical regulatory mechanism of TC-NER in restoring gene expression