Title: dRnh1-GFP: A novel method for detection of R-loops in S. cerevisiae
Abstract:
R-loops, also known as RNA/DNA hybrids, are a naturally occurring and usually transient phenomenon that occur in within the genome. However, errors in RNA processing, DNA repair, and the occurrence of transcription-replication conflicts can lead to extended tracts of R-loops that cause DNA damage. Recent studies have revealed the importance of understanding both the programmed and detrimental aspects of R-loops. Unfortunately, the field is currently hampered by a lack of tools to study R-loops, especially tools that leverage some of the advantages of experimental organisms. Here we report on the development and testing of a novel method for R-loop detection method in S. cerevisiae. The ablation of key catalytic residues of the RNA/DNA hybrid-degrading enzyme RnaseH1 (rnh1) causes the protein to bind R-loops without degrading them, thereby turning it into an R-loop detector. Our so-called dRnh1-GFP construct colocalizes with R-loops and can detect both drug-induced and mutation-induced changes in R-loop levels. This method addresses many of the issues with current methods of R-loop detection as it is inexpensive, fast, able to be conducted in living cells, and is adaptable for high-throughput studies.
Schwacha Lab
Friday, May 17th, 2024
12:00PM
Langley A219B