Organization of core mitochondrial replication components into multiphasic condensates

Genomes are organized across several hierarchical levels. Intracellular phase transitions compartmentalize many genomic processes, including transcription and DNA repair. However, little is known regarding how phase transitions contribute to replication, in which long strands of double- and single-stranded DNA need to be coordinated. Here, we investigated the molecular interactions driving the condensation of core mitochondrial replication components into mt-nucleoids. Complex phase behavior emerged among purified mt-replication components: each nucleic acid colocalized with its cognate architectural protein within multiphasic condensates. Using single-molecule experiments, we found that formation of ssDNA increased the partitioning of its cognate protein mtSSB within the condensate, consistent with the preferential localization of mtSSB to replicating mt-nucleoids. To develop mechanistic insights, we built a minimalistic coarse-grained model of mt-replication components that showed how interactions between binary pairs dictate their assembly within condensates. The multiphasic organization of mt-nucleoids has implications for how replication can be spontaneously organized in cells. HighlightsO_LIComplex co-phase behavior of TFAM-mtDNA depends on their affinity and DNA length C_LIO_LIMt-replication architectural proteins segregate ssDNA and dsDNA within condensates C_LIO_LImtSSB selectively partitions into actively replicating nucleoids in vivo C_LIO_LIExposure of ssDNA promotes mtSSB partitioning into TFAM-mtDNA condensates C_LI