Live cell transcription-coupled nucleotide excision repair dynamics revisited

Diana A. Llerena Schiffmacher, Katarzyna W. Kliza, Arjan F. Theil, Gert Jan Kremers, Jeroen A.A. Demmers, Tomoo Ogi, Michiel Vermeulen, Wim Vermeulen*, Alex Pines*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)
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Abstract

Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags.

Original languageEnglish
Article number103566
Number of pages15
JournalDNA Repair
Volume130
DOIs
Publication statusE-pub ahead of print - 14 Sept 2023

Bibliographical note

Funding Information:
This work was supported by the CW-TOP grant ( 714.017.003 ) and Oncode Institute (partly financed by the Dutch Cancer Society ). We thank the Josephine Nefkens Cancer Program for infrastructural support. We thank Dr. Hannes Lans and Prof dr. Jurgen Marteijn for the constructive discussion.

Publisher Copyright:
© 2023 The Authors

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