Abstract 2472 Firing of OriL in human mitochondrial DNA replication is regulated by nucleic acid- and polymerase-specific effects

Replication of the human mitochondrial DNA chromosome is unique. There are two origins that direct initiation of DNA replication, one for each strand. These origins fire asynchronously and are regulated by different cis-acting elements and trans-acting factors. The regulation of origin firing as well as the fundamental mechanisms of protein recruitment, primer synthesis, handoff, and extension during these processes are still poorly understood. The DNA replication origin of the so-called Light strand, OriL, is comprised of a simple stem-loop structure that forms upon displacement of the parental Heavy strand. This stem-loop is recognized by the mitochondrial RNA polymerase, Polrmt, which catalyzes synthesis of short RNA olignucleotides. These short RNA products, synthesized by Polrmt at OriL, are subsequently extended by the mitochondrial replicative DNA polymerase, Pol Gamma. We use in vitro RNA synthesis assays coupled with mass photometry to probe how protein-nucleic acid and protein-protein interactions orchestrate origin firing at OriL.