The role of RNA polymerase I in ribosomal DNA protection against UV light-induced DNA damage

1 p.DNA lesions threaten cell life and must be repaired to maintain genome integrity. During transcription, RNA polymerases actively scan DNA to find lesions and trigger their repair. In growing eukaryotic cells, about 60% of the total transcriptional activity involves the synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I), a 14-subunit macromolecular machine with unique regulatory features. Accordingly, transcribing Pol I monitors ribosomal DNA (rDNA) integrity and influences cell survival, but how this enzyme handles DNA lesions remains largely unknown. We used cryo-EM and in vitro transcription tests to investigate Pol I transcriptional stalling by one of the most common UV light-induced lesions, i.e. cyclobutane pyrimidine dimers (CPD) [3]. A two-step mechanism operates in Pol I to firmly stall the enzyme at CPD lesions, whereas RNA polymerase II (Pol II), which scans for damage within protein-coding genes, is able to bypass such lesions. First, bridge helix residue Arg1015, which is unique in Pol I, contacts the lesion and significantly reduces the bypass rate, as we confirm by mutational analysis. Second, the intrinsic RNA cleavage activity of Pol I, which in Pol II requires binding of transcription factor IIS (TFIIS), removes RNA nucleotides opposite the lesion. Our results suggest that this dual mechanism strongly blocks rRNA synthesis to hamper incorporation of mutations into ribosomes. These findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.Peer reviewe