Genome-Wide Profiling of Endogenous Single-Stranded DNA Using the SSiNGLe-P1 Method

Endogenous single-stranded DNA (essDNA) can form in a mammalian genome as the result of a variety of molecular processes and can both play important roles inside the cell as well as have detrimental consequences to genome integrity, much of which remains to be fully understood. Here, we established the SSiNGLe-P1 approach based on limited digestion by P1 endonuclease for high-throughput genome-wide identification of essDNA regions. We applied this method to profile essDNA in both human mitochondrial and nuclear genomes. In the mitochondrial genome, the profiles of essDNA provide new evidence to support the strand-displacement model of mitochondrial DNA replication. In the nuclear genome, essDNA regions were found to be enriched in certain types of functional genomic elements, particularly, the origins of DNA replication, R-loops, and to a lesser degree, in promoters. Furthermore, interestingly, many of the essDNA regions identified by SSiNGLe-P1 have not been annotated and thus could represent yet unknown functional elements.

Automated summary

Endogenous single-stranded DNA (essDNA) can form in a mammalian genome and can have both important roles and detrimental consequences to genome integrity. We developed the SSiNGLe-P1 approach to identify essDNA regions in a high-throughput manner. Using this method, we found new evidence for the strand-displacement model in mitochondrial DNA replication and identified functional elements in the nuclear genome that are enriched in essDNA regions, including DNA replication origins, R-loops, and promoters. Additionally, many of the essDNA regions identified have not been annotated and could represent unknown functional elements.