RNA polymerase II contributes to preventing transcription-mediated replication fork stalls

• Published in: The EMBO journal 2015-01, Vol.34 (2), p.236-250
• Main Authors: Felipe-Abrio, Irene, Lafuente-Barquero, Juan, García-Rubio, María L, Aguilera, Andrés
• Format: Article
• Language: English
• Subjects: Biomarkers - metabolism; Chromatin Immunoprecipitation; Deoxyribonucleic acid; DNA; DNA damage; DNA damage response; DNA Repair - genetics; DNA replication; DNA Replication - genetics; double-strand break repair; EMBO09; EMBO13; EMBO44; Gene Expression Profiling; genome instability; Genomic Instability; Genomics; Microbiology; Mutants; Mutation; Mutation - genetics; Oligonucleotide Array Sequence Analysis; Retention; RNA polymerase; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; RNAPII; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - genetics; Transcription factors; Transcription, Genetic; Yeast; Yeasts
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201488544

Transcription is a major contributor to genome instability. A main cause of transcription‐associated instability relies on the capacity of transcription to stall replication. However, we know little of the possible role, if any, of the RNA polymerase (RNAP) in this process. Here, we analyzed 4 specific yeast RNAPII mutants that show different phenotypes of genetic instability including hyper‐recombination, DNA damage sensitivity and/or a strong dependency on double‐strand break repair functions for viability. Three specific alleles of the RNAPII core, rpb1‐1, rpb1‐S751F and rpb9∆, cause a defect in replication fork progression, compensated for by additional origin firing, as the main action responsible for instability. The transcription elongation defects of rpb1‐S751F and rpb9∆ plus our observation that rpb1‐1 causes RNAPII retention on chromatin suggest that RNAPII could participate in facilitating fork progression upon a transcription‐replication encounter. Our results imply that the RNAPII or ancillary factors actively help prevent transcription‐associated genome instability. Synopsis Collisions between RNA transcription and DNA replication machineries impair replication fork (RF) progression and cause DNA breaks, demanding homologous recombination for repair and RF restart. Identification and characterization of specific yeast RNA polymerase II mutations that enhance DNA RF stalling and trigger hyper‐recombination suggests that increased RNAP II retention at transcription sites may contribute to genomic instability and that eukaryotic RNAP II is more than a passive entity in the management of transcription–replication encounters. Specific RNAP II mutants in Saccharomyces cerevisiae , rpb1‐1 , rpb1S71F and rbp9 Δ show an increase in genetic instability as detected by hyper‐recombination, DNA damage sensitivity, and dependency on DSB repair functions for viability. Impaired RF progression in these RNAP II mutants can be compensated by additional origin firing. Under replication stress, checkpoint‐activating DNA lesions accumulate in these mutant cells and are repaired only with considerable delay. RNase H1 overexpression does not suppress genome instability in rpb1 mutants, indicating that an increase in R‐loops is not a main determinant of RF stalling. The causative rpb1‐1 mutation increases retention of RNAP II at the site of transcription. Graphical Abstract Identification of specific yeast RNA polymerase II mutations that cause genomic instability b