Browsing by Author "Haas, Elisha"

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    Inherited Human Sex Reversal due to Loss of a Water-Mediated Hydrogen Bond at a Conserved Protein-DNA Interface
    (Cold Spring Harbor Laboratory Press, 2021) Chen, Yen-Shan; Racca, Joseph; Amir, Dan; Haas, Elisha; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Male sex determination in mammals is initiated by SRY, a Y-encoded architectural transcription factor. The protein contains a high-mobility-group (HMG) box that mediates sequence-specific DNA bending. Mutations in SRY causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box. Although such mutations usually arise de novo in spermatogenesis, some are inherited: male development occurs in one genetic background (the father) but not another (the sterile XY daughter). Here, we compare de novo and inherited mutations at an invariant Tyr adjoining the motif’s basic tail (consensus position 72; Y127C and Y127F in intact SRY). Crystal structures of homologous SOX-DNA complexes suggest that the wild-type side chain’s para-OH group anchors a water-mediated hydrogen bond to the DNA backbone. In an embryonic gonadal cell line, Y127C (de novo) led to accelerated proteasomal proteolysis and blocked transcriptional activity; activity remained low on rescue of expression by chemical proteasome inhibition. Y127F (inherited) preserved substantial transcriptional activity: 91(±11)% on SRY overexpression and 65(±17)% at physiological expression. Control studies indicated no change in protein lifetime or nuclear localization. Only subtle biophysical perturbations were observed in vitro. Although though inherited variant’s specific DNA affinity was only twofold lower than wild type, stopped-flow kinetic analysis revealed a sevenfold decrease in lifetime of the complex. Time-resolved fluorescence energy transfer (using a 15-base pair DNA site) demonstrated native mean DNA bending but with a slightly widened distribution of end-to-end DNA distances. Our findings highlight the contribution of a single water-mediated hydrogen bond to robustness of a genetic switch in human development.
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    Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity
    (Frontiers, 2022-12) Racca, Joseph D.; Chatterjee, Deepak; Chen, Yen-Shan; Rai, Ratan K.; Yang, Yanwu; Georgiadis, Millie M.; Haas, Elisha; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain’s minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father’s male development, but at odds with the daughter’s XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably “unclamps” its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.