The Origin Recognition Complex requires chromatin tethering by a hypervariable intrinsically disordered region that is functionally conserved from sponge to man

Abstract

The first step towards eukaryotic genome duplication is loading of the replicative helicase, the Mcm2-7 complex, onto chromatin. This so-called "licensing" step is executed by the Pre- Replication Complex (Pre-RC) whose assembly on chromatin is nucleated by the DNA-binding activity of the Origin Recognition Complex (ORC). It is thought that metazoan ORC, like the yeast complex, is recruited directly to chromatin by its ATP-dependent DNA binding and encirclement activity. However, we have previously shown that this DNA binding mode is dispensable for chromatin recruitment of fly ORC, raising the question of how metazoan ORC binds chromosomes. We show here that the intrinsically disordered region (IDR) of fly Orc1 is both necessary and sufficient for recruitment of ORC to chromosomes in vivo and demonstrate that this activity is regulated by IDR phosphorylation. In vitro studies show that the IDR alone binds DNA and this bestows the ORC holocomplex with a high-affinity ATP-independent DNA binding mode. Interestingly, we find that Orc1 IDRs have diverged so markedly across metazoans that they are unrecognizable as orthologs and yet we find that these compositionally homologous sequences retain DNA and chromatin binding activity down to basal metazoans. Altogether, these data suggest that chromatin is recalcitrant to ORC's ATP-dependent DNA binding activity and we propose that this necessitates IDR-dependent chromatin tethering which poises ORC to opportunistically encircle nucleosome free regions as they become available. This work reveals a novel step in metazoan replication licensing and expands our understanding of disordered protein homology and evolution by stretching the relationship between primary structure and function.