AlphaFold-guided phylogenetic analyses suggest surprising heterogeneity in metazoan replication origin licensing...

DNA replication initiation is a tightly regulated process that requires the coordinated assembly of replication machineries throughout the genome. During the first step of initiation, origin licensing, the MCM replicative helicase motor is loaded onto replication origins by the origin recognition complex (ORC) as a head-to-head double hexamer complex. Distinct mechanisms have been proposed to facilitate human MCM double hexamer loading, but the physiological relevance of each of them remains unclear. Here, we investigate the evolutionary conservation of these pathways using an AlphaFold-guided structural phylogenetics approach. Our analyses reveal that ORC6, a subunit of ORC previously thought to be essential for origin licensing in vivo, has been lost in multiple metazoan lineages. Despite this loss, many of these species retain an element in ORC3, the ORC3 tether, that can interact with MCM and facilitate an ORC6-independent MCM loading mechanism. AlphaFold2 Multimer predictions suggest that ORC3 tether interactions with MCM are broadly conserved across Metazoa. Our findings support the physiological relevance of ORC6-independent MCM loading, provide experimentally testable hypotheses on origin licensing mechanisms in diverse metazoan species, and highlight how AlphaFold can be leveraged to investigate protein evolution and function over large timescales.