RNase H1 Hybrid-Binding Domain-Based Tools for Cellular Biology Studies of DNA–RNA Hybrids in Mammalian Cells

AbstractR 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.Key wordsDNA–RNA hybridsR loopsRNase H1Hybrid-binding domain (HBD)DNA double-strand breaks (DSB) GFP