Stimulation of direct-repeat recombination by RNA polymerase III transcription

• Published in: DNA repair 2009-05, Vol.8 (5), p.620-626
• Main Authors: de la Loza, M.C. Díaz, Wellinger, R.E., Aguilera, A.
• Format: Article
• Language: English
• Subjects: 4-Nitroquinoline-1-oxide - pharmacology; 4-NQO; Bacteriology; Biological and medical sciences; Blotting, Northern; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Replication - genetics; DNA, Fungal - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Fungal; Genic rearrangement. Recombination. Transposable element; Growth, nutrition, cell differenciation; Microbiology; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Quinolones - pharmacology; Recombination, Genetic; Repetitive Sequences, Nucleic Acid - genetics; RFB; RNA Polymerase III - genetics; RNA, Transfer - genetics; rrm3Δ; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Transcription, Genetic - genetics; Transcription-associated recombination; tRNA SUP53
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2008.12.010

Eukaryotic cells have to regulate the progression and integrity of DNA replication forks through concomitantly transcribed genes. A transcription-dependent increase of recombination within protein-coding and ribosomal genes of eukaryotic cells is well documented. Here we addressed whether tRNA transcription and tRNA-dependent transcription-associated replication pausing leads to genetic instability. Thus, we designed a plasmid based, LEU2 direct-repeat containing system for the analysis of factors that contribute to tRNA SUP53 -dependent genetic instability. We show that tRNA SUP53 transcription is recombinogenic and that recombination can be further stimulated by deletion of the 5′ to 3′ helicase Rrm3. Furthermore, tRNA SUP53 -dependent recombination was markedly increased in the presence of 4-NQO in rrm3Δ cells only. The frequency of recombination events mediated by tRNA SUP53 transcription does not correlate with the appearance and intensity of replication fork pausing sites. Our results provide evidence that the convergent encounter of replication and RNA polymerase III transcription machineries stimulates recombination, although to a lesser extent than RNA polymerase I or II transcription. However, there is no correlation between recombination and the specific replication fork pausing sites found at the tRNA SUP53 gene. Our results indicate that tRNA-specific replication fork pausing sites are poorly recombinogenic.