Replication licensing regulated by an intrinsically disordered region of origin recognition complex

In eukaryotes, the hetero-hexameric origin recognition complex (ORC) is responsible for assembling Mcm2-7 complex into a head-to-head double hexamer (DH), forming pre-replicative complex (pre-RC) that licenses origin DNA for replication initiation. This process is tightly controlled throughout the cell cycle to ensure accurate duplication of the genome. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit plays a critical role in promoting pre-RC assembly. We found that removing a short segment (residues 175-200) from Orc2-IDR or mutating a key isoleucine (194) in this region significantly inhibits replication initiation across the genome and causes cell death. Although the Orc2-IDR mutants can still assemble the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate on DNA, the assembled mutant OCCM exhibits impaired ATP hydrolysis, preventing its conversion into Mcm2-7-ORC (MO) complex and subsequent DH formation. Interestingly, our in vitro assays showed that adding the Orc2-IDR peptide in the pre-RC reactions can rescue this defect. Furthermore, phosphorylation of this Orc2-IDR motif by S-cyclin dependent kinase (S-CDK) blocks its binding to Mcm2, leading to defective pre-RC assembly. Our findings provide crucial mechanistic insights into the multifaceted roles of ORC in modulating MCM loading to support origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase. To gain insights into the underlying mechanism of replication initiation defects associated with Orc2-NTD mutants, we utilized a synchronization-sequencing (syn-seq) assay to examine the broader context of chromatin replication. Syn-seq is a whole-genome sequencing (WGS) method used to measure the efficiency of origin firing. In this method, the genome is divided into small segments, and the sequencing data of the relevant genome isolated at a particular time point provides of the exact copy number of each segment during DNA replication. We included the following three strains in this analysis: ORC2-WT, orc2∆N200, and orc2-I194E. We generated two datasets for each strain at 0 min (G1-phase) and 30 min (S-phase), representing non-replicative and replicative cells, respectively. We then plotted the ratio of copy number between replicative and non-replicative ones against the genomic locus to obtain a genome-wide landscape of replication initiation.