Updated project metadata.
Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the physical stalling of RNA polymerase II by a DNA damage, which triggers the assembly of TC-NER-specific proteins, including CSA, a WD40 domain containing protein, that forms together with DDB1, CUL4A/B and RBX1, a cullin-RING ubiquitin ligase complex (CRL4CSA). Although ubiquitination of several TC-NER proteins by CSA has been reported, it is still unknown how this complex functions in TC-NER progression. To unravel the molecular mechanism underlying TC-NER, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates the step-wise ubiquitination during TC-NER and is required for efficient progression of this process by controlling the dynamic turnover of CRL4CSA complex.