R-Loops and Mitochondrial DNA Metabolism

R-loops forming inadvertently during transcription can threaten genome stability, but R-loops are also formed intentionally, as a means of regulating transcription and other aspects of DNA metabolism. The study of R-loops in mitochondria is in its infancy, and yet there is already clear evidence that they are predominantly located in the major regulatory region of the mammalian mitochondrial genome. Here, we describe how mitochondrial R-loops have been characterized to date, with the emphasis on the problems of their being extremely labile, and how to minimize their loss during extraction. The oft-overlooked issues of RNA-DNA hybrids not being synonymous with R-loops, and adventitious RNA hybridization to DNA, are tackled head on; and possible new approaches are described and placed in the context of future research lines that could reveal the detailed roles of R-loops in the metabolism of mitochondrial DNA.