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.
Bibliographical noteFunding Information:
The authors acknowledge Jean-Marc Egly and Rick Wood for their generous gift of TFIIH and XPA antibodies, respectively, and the genomics core research facility at the faculty of medicine in the Hebrew University for technical advice. Tom Misteli provided LacR-NLS plasmid, Tomoo Ogi provided UVSSA-deficient KPS3-hTERT cells, Susan Janicki provided U2OS 2-6-3 cells. This work was funded by an LUMC Research Fellowship and an NWO-VIDI grant (ALW.016.161.320) to M.S.L., an ERC starting grant (310913) to A.C.O.V., a Young Investigator Grant from the Dutch Cancer Society (KWF-YIG: 11367) to R.G-P., a Manja Gideon Foundation scholarship to H.G.-B., and Israel Science Foundation grants (1710/17 and 1762/17) administered by the Israeli Academy for Science and humanities and The Israel Cancer Association grant (20191630) to S.A. S.A. is the recipient of the Jacob and Lena Joels memorial fund senior lectureship. J.C.W. was supported by NIH grant HL098316 and is a Howard Hughes Medical Institute (HHMI) Investigator and an American Cancer Society Research Professor. T.E.T.M. was supported by an EMBO Long-term fellowship (ALTF 1316-2016) and an HHMI fellowship of The Jane Coffin Childs Memorial Fund for Medical Research.
© 2020, The Author(s).