Structural basis of human transcription\u2013DNA repair coupling

Goran Kokic,Aleksandar Chernev,Henning Urlaub,Patrick Cramer,Felix R Wagner

doi:10.1038/s41586-021-03906-4

Abstract

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription–repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.

Highlights

When polymerase II (Pol II) encounters a bulky lesion in the template strand, transcription elongation stalls and this triggers TCR5
Following ubiquitylation of Pol II, UV-stimulated scaffold protein A (UVSSA) recruits TFIIH and other factors that are required for nucleotide excision repair[4,7,17]
Yeast lacks counterparts of CSA and UVSSA, prompting us to study the human TCR mechanism. This is feasible based on the structure of the Pol II elongation complex EC*, which contains the elongation factors DSIF20, PAF1 complex (PAF), RTF1 and SPT6

Summary

EC–DSIF c

Proteins[28] and yeast Spt[6] binds to DNA29. Modelling shows that extension of upstream DNA leads to a clash with parts of SPT6, and changes in the position of DNA or SPT6 are required to accommodate longer DNA (Extended Data Fig 8b). The E3 ligase CRL4CSA polyubiquitylates CSB, leading to degradation of CSB12 To investigate these events in vitro, we performed ubiquitylation assays with the complete Pol II–TCR complex containing CRL4CSA (Fig. 3a). Ubiquitylation of Pol II was dependent on CSB and occurred in the absence of UVSSA (Extended Data Fig 9a), as shown in vivo[33] These results indicate that CRL4CSA is the E3 ligase that ubiquitylates K1268. In the second state of the complete Pol II–TCR complex structure (structure 5), CUL4A and RBX1 have moved over a large distance to reach the C-terminal region of CSB (Fig. 3c). This CSB region is targeted by ubiquitylation[35] and is essential for TCR36. Conversion of the first state to the second state of the complete ECTCR complex requires extensive rearrangements within CRL4CSA, including an a kDa

Ubiquitin RBX1

Methods