Humanized yeast ORC drives R-loop dependent transcription-replication conflicts and global gene dysregulation

The Origin Recognition Complex (ORC) recruits Mini-Chromosome Maintenance (MCM) hexamers to initiate eukaryotic DNA replication. While yeast ORC binds specific DNA motifs, human ORC binds sequence-agnostically nucleosome-depleted promoter regions. Here, using a mutant yeast model that recapitulates the human ORC binding landscape, we demonstrate that pervasive and unscheduled ORC binding at promoters in the compact yeast genome generates R-loop associated transcription-replication conflicts and disrupts gene expression across thousands of genes. Unexpectedly, we also uncover that substituting endogenous gene promoters with a replication origin sequence maintains robust transcription. We reveal that this intrinsic, promoter-like function relies on the ability of origins to form a nucleosome-depleted region (NDR) encoded within the origin’s sequence composition. However, origin-substituted loci reposition the +1 nucleosome which drives the transcription machinery to select alternative transcription start sites (TSSs). Thus, origins and promoters share an intrinsic chromatin structure-controlled ability to drive transcription initiation. Together, our results show that origin licensing in compact genomes relies on sequence-specific, coupled origin licensing to insulate transcription from the replication machinery.