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

Yana Van Der Weegen,Hadar Golan-Berman,Diana Van Den Heuvel,Sheera Adar,Román González-Prieto,Joachim Goedhart,Martijn S Luijsterburg,Elisheva E Heilbrun,Katja Apelt,Alfred C O Vertegaal,Tycho E T Mevissen,Johannes C Walter

doi:10.1038/s41467-020-15903-8

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.

Highlights

Nucleotide excision repair (NER) is a versatile DNA repair pathway that removes a wide range of helix-distorting
Genomic DNA fragments immunoprecipitated together with RNAPIIo after UV irradiation were highly enriched for the most abundant UV-induced DNA lesion (Fig. 1c). These findings suggest that a considerable fraction of the RNAPIIo molecules we capture under our conditions are stalled at DNA lesions
Our data reveals a sequential and cooperative assembly mechanism of the human Transcription-coupled repair (TCR) complex, which involves the stepwise assembly of CSB, CSA, and UVSSA to target the transcription factor IIH (TFIIH) complex to DNA damage-stalled RNAPIIo to initiate DNA repair (Fig. 7c). It has been recognized for some time that CSA, CSB, and UVSSA are required for TCR, remarkably little is known about how these proteins cooperate to trigger TCR

Summary

Introduction

Nucleotide excision repair (NER) is a versatile DNA repair pathway that removes a wide range of helix-distorting. Transcription-coupled repair (TCR) is a specialized NER sub-pathway that removes DNA lesions from actively transcribed DNA strands[1]. It is believed that the TCR pathway is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions, which triggers the recruitment of the core NER machinery to repair these lesions[2]. The transcription factor IIH (TFIIH) complex is recruited to unwind the DNA3,4, followed by dual incision, and the release of a 22–30 nucleotide-long DNA strand containing the lesion[5,6]. Inherited defects that selectively impair TCR give rise to Cockayne Syndrome (CS) and UV-sensitive syndrome (UVSS). Live-cell imaging suggests that CSB monitors the progression of transcription elongation by continuously probing RNAPIIo complexes[21]. The association of CSB with RNAPII is sufficient to recruit TFIIH in vitro[23], it is unknown whether additional factors are required to trigger the recruitment of the repair machinery in vivo

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Conclusion