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

## TL;DR

This study uses AlphaFold predictions to show that DNA replication mechanisms in animals are more diverse than previously thought, with some species not needing a key protein for replication initiation.

## Contribution

The study reveals that ORC6-independent MCM loading is evolutionarily conserved and functionally viable in multiple metazoan lineages.

## Key findings

- ORC6, once thought essential for origin licensing, is absent in multiple metazoan lineages.
- The ORC3 tether supports MCM loading in the absence of ORC6 and is conserved across Metazoa.
- AlphaFold2 Multimer predictions reveal structural conservation of MCM interactions with ORC3.

## Abstract

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.

The loading of two opposing MCM complexes onto DNA by the initiator ORC (origin recognition complex) constitutes the first step in eukaryotic DNA replication initiation. By integrating AlphaFold structure predictions into phylogenetic analyses, this study shows that flexibility in MCM loading mechanisms, recently discovered through in vitro reconstitution of human MCM loading, is common across Metazoa.

Some metazoan taxa have lost ORC’s ORC6 subunit, indicating that ORC6-independent MCM loading naturally occurs in Metazoa.Loss of ORC6 is accompanied by structural and functional degeneracy of the ORC6-binding site in ORC.The ORC3 tether, a structural element that can support MCM-ORC intermediate assembly without ORC6, and its interaction with MCM2, are broadly conserved across Metazoa.Systematic AlphaFold structure predictions of protein orthologs constitute a powerful approach to examine protein conservation and function across large evolutionary timescales.

Some metazoan taxa have lost ORC’s ORC6 subunit, indicating that ORC6-independent MCM loading naturally occurs in Metazoa.

Loss of ORC6 is accompanied by structural and functional degeneracy of the ORC6-binding site in ORC.

The ORC3 tether, a structural element that can support MCM-ORC intermediate assembly without ORC6, and its interaction with MCM2, are broadly conserved across Metazoa.

Systematic AlphaFold structure predictions of protein orthologs constitute a powerful approach to examine protein conservation and function across large evolutionary timescales.

Flexibility regarding ORC6-dependent or -independent MCM loading by ORC appears to be common across Metazoa.