Abstract

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the rRNA precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes abasic DNA lesions. Here, we report electron cryo-microscopy (cryo-EM) structures of Pol I at different stages of stalling at abasic sites, supported by in vitro transcription studies. Our results show that templating abasic sites can slow nucleotide addition by base sandwiching between the RNA 3-end and the Pol I bridge helix. However, the presence of a templating abasic site induces opening of the Pol I cleft for either enzyme dissociation from DNA or for access of A12-Ct into the active site to stimulate RNA cleavage. Nucleotide addition opposite the lesion induces an early translocation intermediate that is different from previously-described RNA polymerase paused states, as DNA bases in the hybrid tilt to form hydrogen bonds with the newly-added RNA base. While in this state nucleotide addition is strongly disfavoured, intrinsic Pol I RNA cleavage activity acts as a failsafe mechanism to minimize lesion bypass. Our results uncover a two-step mechanism leading to persistent Pol I stalling after nucleotide addition opposite Ap sites, which is distinct from arrest by CPD lesions and from Pol II blockage at Ap sites.