Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of... Show moreCells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.Cells employ global genome nucleotide excision repair to repair a broad spectrum of genomic DNA lesions. Here, the authors reveal how chromatin is primed for repair, providing insight into mechanisms of chromatin plasticity during DNA repair. Show less
Weegen, Y. van der; Lint, K. de; Heuvel, D. van den; Nakazawa, Y.; Mevissen, T.E.T.; Schie, J.J.M. van; ... ; Luijsterburg, M.S. 2021
Two side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.Cells employ transcription-coupled repair (TCR) to... Show moreTwo side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4(CSA) ubiquitin ligase. How CRL4(CSA) is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4(CSA) for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication. Show less
Heuvel, D. van den; Weegen, Y. van der; Boer, D.E.C.; Ogi, T.; Luijsterburg, M.S. 2021
DNA lesions pose a major obstacle during gene transcription by RNA polymerase II (RNAPII) enzymes. The transcription-coupled DNA repair (TCR) pathway eliminates such DNA lesions. Inherited defects... Show moreDNA lesions pose a major obstacle during gene transcription by RNA polymerase II (RNAPII) enzymes. The transcription-coupled DNA repair (TCR) pathway eliminates such DNA lesions. Inherited defects in TCR cause severe clinical syndromes, including Cockayne syndrome (CS). The molecular mechanism of TCR and the molecular origin of CS have long remained enigmatic. Here we explore new advances in our understanding of how TCR complexes assemble through cooperative interactions between repair factors stimulated by RNAPII ubiquitylation. Mounting evidence suggests that RNAPII ubiquitylation activates TCR complex assembly during repair and, in parallel, promotes processing and degradation of RNAPII to prevent prolonged stalling. The fate of stalled RNAPII is therefore emerging as a crucial link between TCR and associated human diseases. Show less
The lysosomal β-glucosidase named glucocerebrosidase (GCase) is a retaining β-glucosidase that hydrolyzes the glycosphingolipid glucosylceramide (GlcCer) to ceramide and glucose at acid pH.... Show moreThe lysosomal β-glucosidase named glucocerebrosidase (GCase) is a retaining β-glucosidase that hydrolyzes the glycosphingolipid glucosylceramide (GlcCer) to ceramide and glucose at acid pH. Inherited deficiency of GCase causes Gaucher disease (GD), a relatively common lysosomal storage disorder. GCase fulfills another crucial function beyond lysosomes. The enzyme generates ceramides from GlcCer molecules in the outer part of the skin, the stratum corneum. This is essential for skin barrier properties compatible with terrestrial life. GCase is catalytically versatile and can hydrolyze as well as catalyze transglycosylation.In this thesis a novel sensitive in situ method for the detection of active GCase in skin sections is described. Followed by a study of skin sections of patiens with atopic dermatitis revealing that the localization and activity of GCase and acid sphingomyelinase (ASM) was abnormal in skin of AD patients, particularly at lesional skin sites.It is demonstrated that GCase not only cleaves 4-methylumbelliferyl-β-D-glucose, but also 4-methylumbelliferyl-β-D-xylose. It is reported for the first time that GCase is able to transxylosylate cholesterol to render xylosyl-β-cholesterol (XylChol). The formed XylChol can act as a subsequent acceptor for further transxylosylation, rendering di-xylosyl-cholesterol. And finally the discovery of of GlcChol as novel component of human epidermis is reported. Show less
Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside... Show moreDeficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as acceptor for subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced β-glucosidase activity were similarly impaired in β-xylosidase, transglucosidase and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from Gaucher disease patients. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyse formation of XylCer. Thus, food-derived β-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids. Show less
The existence of glucosylated cholesterol (GlcChol) in tissue has recently been recognized. GlcChol is generated from glucosylceramide (GlcCer) and cholesterol through transglucosylation by two... Show moreThe existence of glucosylated cholesterol (GlcChol) in tissue has recently been recognized. GlcChol is generated from glucosylceramide (GlcCer) and cholesterol through transglucosylation by two retaining β-glucosidases, GBA and GBA2. Given the abundance of GBA, GlcCer and cholesterol in the skin's stratum corneum (SC), we studied the occurrence of GlcChol. A significant amount of GlcChol was detected in SC (6 pmol/mg weight). The ratio GlcChol/GlcCer is higher in SC than epidermis, 0.083 and 0.011, respectively. Examination of GlcChol in patients with Netherton syndrome revealed comparable levels (11 pmol/mg). Concluding, GlcChol was identified as a novel component in SC and is likely locally metabolized by GBA. The physiological function of GlcChol in the SC warrants future investigation. Show less
Aerts, J.M.F.G.; Kuo, C.L.; Lelieveld, L.T.; Boer, D.E.C.; Lienden, M.J.C. van der; Overkleeft, H.S.; Artola, M. 2019
Glycosphingolipids are important building blocks of the outer leaflet of the cell membrane. They are continuously recycled, involving fragmentation inside lysosomes by glycosidases. Inherited... Show moreGlycosphingolipids are important building blocks of the outer leaflet of the cell membrane. They are continuously recycled, involving fragmentation inside lysosomes by glycosidases. Inherited defects in degradation cause lysosomal glycosphingolipid storage disorders. The relatively common glycosphingolipidosis Gaucher disease is highlighted here to discuss new insights in the molecular basis and pathophysiology of glycosphingolipidoses reached by fundamental research increasingly using chemical biology tools. We discuss improvements in the detection of glycosphingolipid metabolites by mass spectrometry and review new developments in laboratory diagnosis and disease monitoring as well as therapeutic interventions. Show less
