Transcriptional R-loops in Archaea

Primary author: Marguerite Smith
Co-author(s): Michael Rolfsmeier; Cynthia Haseltine
Faculty sponsor: Cynthia Haseltine

Primary college/unit: College of Veterinary Medicine
Campus: Pullman


To maintain genome integrity, cells must contend with multiple DNA structures that arise during replication, transcription, and repair. Transcriptionally active regions of the genome are particularly susceptible to DNA damage through the production of R-loop regions, consisting of an RNA:DNA hybrid along with a displaced ssDNA non-template strand, which is particularly susceptible to damage. Failure to dissociate RNA:DNA hybrids can lead to extensive ssDNA regions, stalled transcription machinery, or persistent protein:DNA complexes, all of which are potentially deleterious to the cell. Thus, all organisms must have robust mechanisms to contend with RNA:DNA hybrids within actively transcribed regions of the genome. While transcriptionally associated R-loops have been detected in bacteria and eukaryotes, there have been no reports of their occurrence in archaea. We used the hyperthermophilic crenarchaeon Sulfolobus solfataricus as a model archaeal species to investigate the formation of RNA:DNA hybrids. We established methodology for immunoprecipitation of RNA:DNA hybrids from S. solfataricus and evaluated abundance and persistence of these hybrids for both transcriptionally active and inactive regions of the genome using multiple cell backgrounds. In parallel, we examined topological alterations in the supercoiling state of a plasmid carrying an inducible gene using an in vitro transcription approach. Our molecular characterization of RNA:DNA hybrids in S. solfataricus indicates they are strongly associated with transcriptionally active regions and likely represent R-loop structures. Additionally, variation of production and persistence of these R-loops in strain backgrounds with differential DNA damage responses implicates DSB repair processes in maintaining genome stability during transcriptional activities in S. solfataricus.