The CRYO-EM structure of RNA polymerase I stalled at UV light-induced damage unravels a new molecular mechanism to identify lesions on ribosomal DNA

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-10-2019In eukaryotic cells, three RNA polymerases transcribe the genome, each specialized in transcribing a specific set of genes. Pol II synthesizes mRNA, Pol III produces short untranslated RNAs and Pol I transcribes ribosomal DNA (rDNA). The latter produces the rRNA precursor, which after maturation constitutes the backbone of the ribosome. Pol I accounts for approximately 60% of the total transcriptional activity in growing cells and also carries out the supervision of rDNA integrity. Therefore, it is a key determinant for the control of the normal function of the cell. Environmental threats can generate DNA lesions that are cytotoxic for the cell and one of the most known is UV-light. The principal DNA damage produced by this external agent is cis-syn cyclobutane pyrimidine dimers (CPDs), a bulky DNA lesion that can introduce distortions in the DNA helix, thus obstructing fundamental processes such as transcription. The main goal of this Ph.D. Thesis is understanding the structural basis of Pol I stalled at UV light-induced DNA damage. The principal contribution is the cryo-EM structure at 3.6 Å resolution and the derived atomic model of Pol I in elongation complex containing a CPD lesion at the DNA TS. This structure shows that the CPD lesion induces an early translocation intermediate, along with several conformational rearrangements in Pol I structural elements inside the DNA binding cleft, which contribute to enzyme stalling. The structure revealed that the BH residue R1015 plays a relevant role for enzyme arresting, which was confirmed by mutational analysis using E.coli RNA polymerase as a model system. In vitro transcription assays comparing the Pol I and Pol II behavior in the presence of CPD reveal that, while Pol II can slowly bypass the lesion, Pol I stalls right before the lesion due to the balance between a slow nucleotide incorporation and a fast-intrinsic RNA cleavage activity. Altogether, our results reveal the molecular mechanism of Pol I stalling at CPD lesions, which is distinct from Pol II arrest. This PhD Thesis opens the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNATesis realizada gracias a la ayuda BES-2014-070708 del Ministerio de Ciencia, Innovación y Universidade