Humanizing the Yeast Origin Recognition Complex

The evolutionarily conserved Origin Recognition Complex (ORC), plays a key role in origin selection in eukaryotes. However, ORC is strikingly divergent in its DNA binding specificity, ranging from base-specific interactions in Saccharomyces cerevisiae to base-agnostic interactions in humans. The mechanisms underlying this distinct selectivity is unknown. Atomic model of the yeast ORC showed that base-specific interaction with the invariant thymines of the ARS consensus sequence (ACS) is encoded by a 19-amino acid insertion helix (IH) embedded in the winged helix domain (WHD) of Orc4. This IH is absent in the Orc4 of metazoans including humans, suggesting that removal of the IH might give the yeast ORC “human-like” properties. Indeed, yeast strain engineered with IH deficient Orc4 has completely altered ORC-binding sites enriched in poly-dT tracts located in larger nucleosome-depleted and intergenic open chromatin. In vivo and in vitro assays show that the mutant ORC loads MCM efficiently, in spite of its altered specificity in favor of binding patterns more characteristic of those observed in humans/metazoans. This work provides insights for understanding how ORC evolves to adopt a life cycle that requires plasticity in origin selection during development. 2 replicates for Myc-tag ORC ChIP-seq for G2/M arrested WT and mutant yeast cells. 2 replicates for FLAG-tag MCM ChIP-seq for G1 arrested WT and mutant yeast. 1 replicate for BrdU IP-seq for early S arrested WT and mutant yeast cells. 1 replicate for MNase-seq for G1 arrested WT and mutant yeast cells. 1 replicate for ATAC-seq for G2M arrested WT and mutant yeast cells.