Naveh-Tassa, S. | bioRxiv Science

bioRxiv2024

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.

cell biology↗

bioRxiv2023

Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain

Homochirality of biopolymers emerged early in the history of life on Earth, nearly 4 billion years ago. Whether the establishment of homochirality was the result of abiotic physical and chemical processes, or biological selection, remains unknown. However, given that significant events in protein evolution predate the last universal common ancestor, the history of homochirality may have been written into some of the oldest protein folds. To test this hypothesis, the evolutionary trajectory of the ancient and ubiquitous helix-hairpin-helix (HhH) protein family was analyzed for functional robustness to total chiral inversion of just one binding partner. Against expectations, functional ambidexterity was observed across the entire trajectory, from phase separation of HhH peptides with RNA to dsDNA binding of the duplicated (HhH)2-Fold. Moreover, dissociation kinetics, mutational analysis, and molecular dynamics simulations revealed significant overlap between the binding modes of a natural and a mirror-image protein to natural dsDNA. These data suggest that the veil between worlds with alternative chiral preferences may not be as impenetrable as is often assumed, and that the HhH protein family is an intriguing exception to the dogma of reciprocal chiral substrate specificity proposed by Milton and Kent (Milton et al. Science 1992).

biophysics↗