An Orc1 initiator-specific motif (ISM)-related region limits ORC-ssDNA binding and promotes replication origin...

Single-stranded DNA (ssDNA) is an essential intermediate of genome duplication but can also arise in the genome, including at highly transcribed loci. Although the origin recognition complex (ORC), a eukaryotic replication initiator, has been reported to bind ssDNA, how interactions with ssDNA-exposing regions are regulated without compromising origin specificity and genome stability remains poorly understood. Here, we characterize the ssDNA-binding properties of budding yeast ORC and the role of an initiator-specific motif (ISM)-related region within the AAA+ domain of Orc1. In vitro, ORC binds ssDNA (except for poly dA) at affinities comparable to those of replication protein A (RPA) under identical binding conditions, suggesting base composition-dependent modulation of ORC-ssDNA binding. Genome-wide analyses show partial overlap between ORC- and RPA-enriched peaks, consistent with ORC binding at a subset of ssDNA-forming loci in vivo. Mutations of the Orc1 ISM-related region increase ORC binding to ssDNA and promote higher-order ORC-ssDNA complex formation in vitro, indicating that this region normally limits ORC-ssDNA binding. In contrast, these mutations impair origin-specific ORC binding through mechanisms involving the essential A-element-proximal region. Genetic and chromatin-based analyses further reveal that enhanced ssDNA binding correlates with reduced origin binding in vivo, indicating a redistribution of ORC from replication origins to ssDNA-forming loci. Despite conservation of the ISM across domains of life, these results suggest that eukaryotic ORC has functionally diverged such that origin binding is coupled to the repression of ssDNA binding through the Orc1 ISM-related region, thereby safeguarding faithful origin selection.