The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II

Abstract The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.

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The sequential and cooperative action of CSB, CSA and UVSSA targets the TFIIH complex to DNA damage-stalled RNA polymerase II

10.1101/707216 ◽

2019 ◽

Author(s):

Yana van der Weegen ◽

Hadar Golan Berman ◽

Tycho E.T. Mevissen ◽

Katja Apelt ◽

Román González-Prieto ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cooperative Action ◽

The Core ◽

Assembly Mechanism ◽

Key Factor ◽

Cooperative Assembly

Summary The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings reveal a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.

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A Fully Recombinant System for Activator-dependent Archaeal Transcription

Journal of Biological Chemistry ◽

10.1074/jbc.c400446200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 51719-51721 ◽

Cited By ~ 51

Author(s):

Mohamed Ouhammouch ◽

Finn Werner ◽

Robert O. J. Weinzierl ◽

E. Peter Geiduschek

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Regulators ◽

The Core ◽

General Transcription Factor ◽

Core Components ◽

Archaeal Transcription ◽

Bacterial Type

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from thein vitroassembly of the 12-subunit RNA polymerase from the hyperthermophileMethanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

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DNA damage stabilizes interaction of CSB with the transcription elongation machinery

The Journal of Cell Biology ◽

10.1083/jcb.200401056 ◽

2004 ◽

Vol 166 (1) ◽

pp. 27-36 ◽

Cited By ~ 84

Author(s):

Vincent van den Boom ◽

Elisabetta Citterio ◽

Deborah Hoogstraten ◽

Angelika Zotter ◽

Jean-Marc Egly ◽

...

Keyword(s):

Molecular Weight ◽

Dna Damage ◽

Dna Repair ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

High Molecular Weight ◽

Transcription Elongation ◽

Cockayne Syndrome ◽

Dna Lesions ◽

High Molecular Weight Complex

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

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Human HMGN1 and HMGN2 are not required for transcription-coupled DNA repair

10.1101/835868 ◽

2019 ◽

Author(s):

Katja Apelt ◽

Iris Zoutendijk ◽

Dennis Y. Gout ◽

Diana van den Heuvel ◽

Martijn S. Luijsterburg

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Uv Light ◽

Dna Lesions ◽

Illudin S ◽

Highly Sensitive ◽

Nucleosome Binding ◽

Active Genes

SummaryTranscription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Stalling of RNA polymerase II (RNAPII) at DNA lesions initiates TCR through the recruitment of the CSB and CSA proteins. The full repertoire of proteins required for human TCR – particularly in a chromatin context - remains to be determined. Studies in mice have revealed that the nucleosome-binding protein HMGN1 is required to enhance the repair of UV-induced lesions in transcribed genes. However, whether HMGN1 is required for human TCR remains unaddressed. Here, we show that knockout or knockdown of HMGN1, either alone or in combination with HMGN2, does not render human cells sensitive to UV light or Illudin S-induced transcription-blocking DNA lesions. Moreover, transcription restart after UV irradiation was not impaired in HMGN-deficient cells. In contrast, TCR-deficient cells were highly sensitive to DNA damage and failed to restart transcription. Furthermore, GFP-tagged HMGN1 was not recruited to sites of UV-induced DNA damage under conditions were GFP-CSB readily accumulated. In line with this, HMGN1 did not associate with the TCR complex, nor did TCR proteins require HMGN1 to associate with DNA damage-stalled RNAPII. Together, our findings suggest that HMGN1 and HMGN2 are not required for human TCR.

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