Biology subjects

Adiji, O. A.

Publications and source records attributed to Adiji, O. A..

2 recordsLinked to original sources

bioRxiv2026

Phosphorylation alters the bulk chemical properties of Orc1 to tune DNA binding, phase separation, and heterochromatin partitioning

The first step in initiating DNA replication is binding of the origin recognition complex (ORC) to chromosomes. Metazoan ORC is recruited to chromatin via the Orc1 intrinsically disordered region (IDR) whose DNA and chromatin binding activity are regulated by Cyclin Dependent Kinase (CDK) phosphorylation. ORC is also enriched in heterochromatin where it is required for the formation and maintenance of a silenced chromatin state. ORCs recruitment to heterochromatin is developmentally and cell cycle regulated but the underlying regulatory mechanism remains unknown. We hypothesized that CDK-dependent phosphorylation of the Orc1 IDR underpins regulated recruitment to heterochromatin. Using bioinformatic analyses, we find that the Drosophila Orc1 IDR (Orc1IDR) contains an exceptionally high density of CDK phospho-sites and, despite considerable sequence variation, the density of sites, but not their position, is conserved. In vitro DNA binding and phase separation experiments reveal that phosphorylation tunes Orc1IDR function in a rheostat-like fashion. Using phospho-mimetic variants, we find that constitutive phosphorylation not only weakens interphase chromatin binding but fully inhibits partitioning of Orc1IDR into heterochromatin. Finally, we use phospho-mimetic variants to probe the importance of site-specific phosphorylation and find that the precise position of sites can be changed provided the new sites are equitably distributed across the sequence. These studies demonstrate that phosphorylation tunes the biochemical properties of the Orc1 IDR to control DNA binding, phase separation, and, consequentially, heterochromatin recruitment. This work suggests that localized dephosphorylation of the DNA binding Orc1 IDR may underlie recruitment of ORC to specific genomic loci.

biochemistry↗

bioRxiv2023

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

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 ORCs 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.

biochemistry↗