R loops are abundant noncanonical DNA-RNA hybrid structures that can occur during DNA-based processes, such as transcription, replication and DNA damage, and can lead both to physiologically favorable and pathological outcomes. With an increasing body of work feeding the field of R loop biology, our understanding of the processes in which R loops intervene and the consequences of meddling with R loop formation and dissolution has greatly increased but it has also led to new questions and sometimes opposing possibilities. Proper detection of these structures is a crucial factor to advance our knowledge about R loops and factors associated with their formation and removal. Here, we describe the use of fluorescently tagged HBD, the hybrid-binding domain of RNase H1, as a tool for analyzing DNA-RNA hybrids in different contexts using live-cell microscopy and immunofluorescence experiments.
Keywords: DNA double-strand breaks (DSB); DNA–RNA hybrids; GFP; Hybrid-binding domain (HBD); R loops; RNase H1.
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.