Ordered and disordered regions of the Origin Recognition Complex (ORC) combine to direct in-vivo binding at symmetric and non-symmetric motif sequences.

The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in-vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: an asymmetric motif whose binding requires Orc1-BP4 and Orc4-IH, and a symmetric one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in-vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition.