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.

Dynamic Patterns of Mammalian Mitochondrial DNA Replication Uncovered Using SSiNGLe-5'ES.

The proper replication of mitochondrial DNA is key to the maintenance of this crucial organelle. Multiple studies aimed at understanding the mechanisms of replication of the mitochondrial genome have been conducted in the past several decades; however, while highly informative, they were conducted using relatively low-sensitivity techniques. Here, we established a high-throughput approach based on next-generation sequencing to identify replication start sites with nucleotide-level resolution and applied it to the genome of mitochondria from different human and mouse cell types. We found complex and highly reproducible patterns of mitochondrial initiation sites, both previously annotated and newly discovered in this work, that showed differences among different cell types and species. These results suggest that the patterns of the replication initiation sites are dynamic and might reflect, in some yet unknown ways, the complexities of mitochondrial and cellular physiology. Overall, this work suggests that much remains unknown about the details of mitochondrial DNA replication in different biological states, and the method established here opens up a new avenue in the study of the replication of mitochondrial and potentially other genomes.