STK19 positions TFIIH for cell-free transcription-coupled DNA repair

In transcription-coupled nucleotide excision repair (TC-NER), stalled RNA polymerase II (RNA Pol II) binds CSB and CRL4CSA, which cooperate with UVSSA and ELOF1 to recruit TFIIH. To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also requires STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9-Å cryo-electron microscopy structure shows that STK19 binds the TC-NER complex through CSA and the RPB1 subunit of RNA Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of RNA Pol II for lesion verification. Our analysis of cell-free TC-NER suggests that STK19 couples RNA Pol II stalling to downstream repair events. [Display omitted] •Establishment of a cell-free system for vertebrate transcription-coupled DNA repair•AlphaFold-Multimer screen predicts binding partners of STK19, including TFIIH•Cryo-EM structure of STK19-containing TC-NER complex at 1.9 Å resolution•STK19 couples RNA polymerase II stalling to downstream repair events Together with structural analyses, a cell-free system for transcription-coupled nucleotide excision repair (TC-NER) shows that STK19 is an integral component of the TC-NER complex that couples RNA polymerase II stalling to downstream repair events.