scholarly journals How to limit the speed of a motor: The intricate regulation of the XPB ATPase and Translocase in TFIIH

2020 ◽  
Author(s):  
J. Kappenberger ◽  
W. Koelmel ◽  
E. Schoenwetter ◽  
T. Scheuer ◽  
J. Woerner ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Nucleotide Excision Repair ◽  
Transcription Initiation ◽  
Excision Repair ◽  
Interaction Network ◽  
Correlative Analysis ◽  
General Transcription Factor ◽  
Nucleotide Excision ◽  
Maximum Activation ◽  
Catalytic Functions

AbstractThe superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. In both processes the ATPase activity of XPB is essential. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on a crystal structure of the full p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB’s ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB that acts as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB’s ATPase activity. Our data unravel an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.

scholarly journals How to limit the speed of a motor: the intricate regulation of the XPB ATPase and translocase in TFIIH

2020 ◽  
Vol 48 (21) ◽  
pp. 12282-12296
Author(s):  
Jeannette Kappenberger ◽  
Wolfgang Koelmel ◽  
Elisabeth Schoenwetter ◽  
Tobias Scheuer ◽  
Julia Woerner ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Nucleotide Excision Repair ◽  
Transcription Initiation ◽  
Excision Repair ◽  
Interaction Network ◽  
Correlative Analysis ◽  
General Transcription Factor ◽  
Nucleotide Excision ◽  
Maximum Activation ◽  
Catalytic Functions

Abstract The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB’s ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB’s ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.

scholarly journals Nucleotide excision repair syndromes: molecular basis and clinical symptoms

1995 ◽  
Vol 347 (1319) ◽  
pp. 75-81 ◽  
Keyword(s):  
Nucleotide Excision Repair ◽  
Transcription Initiation ◽  
Clinical Symptoms ◽  
Excision Repair ◽  
Complementation Group ◽  
Molecular Defect ◽  
Cockayne's Syndrome ◽  
Nucleotide Excision ◽  
Group B ◽  
Deficiency Diseases

The phenotypic consequences of a nucleotide excision repair (NER) defect in man are apparent from three distinct inborn diseases characterized by hypersensitivity of the skin to ultraviolet light and a remarkable clinical and genetic heterogeneity. These are the prototype repair syndrome, xeroderma pigmentosum (XP) (seven genetic complementation groups, designated XP-A to XP-G), Cockayne’s syndrome (two groups: CS-A and CS-B) and PIBIDS, a peculiar photosensitive form of the brittle hair disease trichothiodystrophy (TTD, at least two groups of which one equivalent to XP-D). To investigate the mechanism of NER and to resolve the molecular defect in these NER deficiency diseases we have focused on the cloning and characterization of human DNA repair genes. One of the genes that we cloned is ERCC3 . It specifies a chromatin binding helicase. Transfection and microinjection experiments demonstrated that mutations in ERCC3 are responsible for XP complementation group B, a very rare form of XP that is simultaneously associated with Cockayne’s syndrome (CS). The ERCC3 protein was found to be part of a multiprotein complex (TFIIH) required for transcription initiation of most structural genes and for NER . This defines the additional, hitherto unknown vital function of the gene. This ERCC3 gene and several other ner genes involved in transcription initiation will be discussed.

scholarly journals Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin

2020 ◽  
Vol 117 (31) ◽  
pp. 18608-18616 ◽  
Author(s):  
Mingrui Duan ◽  
Kathiresan Selvam ◽  
John J. Wyrick ◽  
Peng Mao
Keyword(s):  
Nucleotide Excision Repair ◽  
Transcription Initiation ◽  
Excision Repair ◽  
Elongation Factor ◽  
Cockayne Syndrome ◽  
Proximal Half ◽  
Genome Wide ◽  
Nucleotide Excision ◽  
Nucleotide Resolution

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion ofSPT4significantly restores TC-NER across the genome in arad26∆mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.

scholarly journals Cryo-EM structure of TFIIH/Rad4–Rad23–Rad33 in damaged DNA opening in nucleotide excision repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Trevor van Eeuwen ◽  
Yoonjung Shim ◽  
Hee Jong Kim ◽  
Tingting Zhao ◽  
Shrabani Basu ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Genomic Dna ◽  
Excision Repair ◽  
Dna Lesions ◽  
Dna Adduct ◽  
Dna Unwinding ◽  
General Transcription Factor ◽  
Nucleotide Excision ◽  
Torque Generation ◽  
Transcription Factor Complex

AbstractThe versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4–Rad23-Rad33 (yeast homologue of XPC–RAD23B–CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9–9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.

scholarly journals Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK

2000 ◽  
Vol 14 (3) ◽  
pp. 349-359 ◽  
Author(s):  
Sofia J. Araújo ◽  
Franck Tirode ◽  
Frederic Coin ◽  
Helmut Pospiech ◽  
Juhani E. Syväoja ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Transcription Initiation ◽  
Kinase Inhibitor ◽  
Excision Repair ◽  
Muscular Atrophy ◽  
Minimal Set ◽  
Repair Synthesis ◽  
Dna Lesion ◽  
Nucleotide Excision ◽  
Recombinant Polypeptides

During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC–hHR23B, XPG, and ERCC1–XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC–hHR23B, XPG, and ERCC1–XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase δ or ε, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.

scholarly journals B Lymphocytes of Xeroderma Pigmentosum or Cockayne Syndrome Patients with Inherited Defects in Nucleotide Excision Repair Are Fully Capable of Somatic Hypermutation of Immunoglobulin Genes

1997 ◽  
Vol 186 (3) ◽  
pp. 413-419 ◽  
Author(s):  
Nayun Kim ◽  
Karen Kage ◽  
Fumihiko Matsuda ◽  
Marie-Paule Lefranc ◽  
Ursula Storb
Keyword(s):  
Dna Repair ◽  
B Cells ◽  
Nucleotide Excision Repair ◽  
Somatic Mutation ◽  
Transcription Initiation ◽  
Somatic Hypermutation ◽  
Excision Repair ◽  
Point Mutations ◽  
Nucleotide Excision ◽  
Gene Switch

Recent experiments have strongly suggested that the process of somatic mutation is linked to transcription initiation. It was postulated that a mutator factor loads onto the RNA polymerase and, during elongation, causes transcriptional arrest that activates DNA repair, thus occasionally causing errors in the DNA sequence. We report the analysis of the role of one of the known DNA repair systems, nucleotide excision repair (NER), in somatic mutation. Epstein–Barrvirus-transformed B cells from patients with defects in NER (XP-B, XP-D, XP-V, and CS-A) were studied. Their heavy and light chain genes show a high frequency of point mutations in the variable (V), but not in the constant (C) regions. This suggests that these B cells can undergo somatic hypermutation despite significant defects in NER. Thus, it is doubtful that NER is an essential part of the mechanism of somatic hypermutation of Ig genes. As an aside, NER seems also not involved in Ig gene switch recombination.

scholarly journals C. elegans TFIIH subunit GTF-2H5/TTDA is a non-essential transcription factor indispensable for DNA repair

2021 ◽  
Author(s):  
Karen L. Thijssen ◽  
Melanie van der Woude ◽  
Carlota Davo-Martinez ◽  
Mariangela Sabatella ◽  
Wim Vermeulen ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Transcription Initiation ◽  
Potential Model ◽  
Excision Repair ◽  
C Elegans ◽  
Nucleotide Excision ◽  
Or Gene ◽  
The Stability ◽  
Essential Transcription Factor

The 10-subunit TFIIH complex is vital to both transcription initiation and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental brittle hair disorder trichothiodystrophy (TTD). Some TTD features are thought to be caused by subtle transcription or gene expression defects. Strikingly, TTDA/GTF2H5 knockout mice are not viable, which makes it difficult to investigate how TTDA/GTF2H5 promotes transcription in vivo. Here, we show that deficiency of the C. elegans TTDA ortholog GTF-2H5 is, however, compatible with viability and growth, in contrast to depletion of other TFIIH subunits. We also show that GTF-2H5 promotes the stability of TFIIH in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of the TFIIH complex to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in the absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as potential model for studying the pathogenesis of this disease.

scholarly journals The role of XPB/Ssl2 dsDNA translocation processivity in transcription-start-site scanning

2020 ◽  
Author(s):  
Eric J. Tomko ◽  
Olivia Luyties ◽  
Jenna K. Rimel ◽  
Chi-Lin Tsai ◽  
Jill O. Fuss ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Atp Hydrolysis ◽  
Excision Repair ◽  
Dna Helicase ◽  
Start Site ◽  
Transcription Start ◽  
Pol Ii ◽  
General Transcription Factor ◽  
Nucleotide Excision

AbstractThe general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates the C-terminal domain to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.

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