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Transcription-blocking DNA 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 stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process. Transcription-Coupled Nucleotide Excision Repair (TC-NER) removes transcription-blocking DNA lesions. This study reveals that DDA1 is a crucial player in TC-NER, protecting transcription programs against genotoxic insults. [ABSTRACT FROM AUTHOR]

For organisms, a fundamental problem is how the huge genome can be searched appropriately for DNA damage in order to prevent genome instability and/or cell death. In mammalian nucleotide excision repair (NER), this process involves multiple protein factors containing the xeroderma pigmentosum gene products (UV-DDB, XPC, TFIIH), which sense different aspects of DNA structural abnormalities, respectively. Coordinated stepwise actions of these factors contribute to simultaneous achievement of versatility, efficiency and accuracy of the DNA quality control for NER. [ABSTRACT FROM AUTHOR]

GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase ∊ (Pol ∊) holoenzyme and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol ∊ is more processive and dissociates more readily from replicated DNA, while under identical conditions, proliferating cell nuclear antigen slightly stimulates Pol ∊ in vitro. These results strongly suggest that GINS is a Pol ∊ accessory protein during chromosomal DNA replication in budding yeast. Based on these results, we propose a model for molecular dynamics at eukaryotic chromosomal replication fork. [ABSTRACT FROM AUTHOR]