Author ORCID Identifier

https://orcid.org/0000-0002-7699-0480

Date of Award

8-10-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Gregory M. K. Poon

Second Advisor

W. David Wilson

Third Advisor

Markus W. Germann

Fourth Advisor

Giovanni Gadda

Abstract

Protein-DNA interactions involve a redistribution of conformational dynamics and hydration properties. As a model DNA-binding protein, structural and thermodynamic studies have shown that ETS transcription factor PU.1 DNA selection is dependent on solvent reorganization. PU.1 high-affinity DNA binding is osmotically sensitive, whereas low-affinity binding is not, which provides a model for examining the role of hydration and DNA dynamics in protein-DNA specificity. Characteristic of ETS members, PU.1 recognizes DNA sequences harboring a central 5’-G0G1A2A3-3’ consensus, where an Arg sidechain contacts O6 of G1 in the major groove. This conserved interaction presented an opportunity to isolate the contributions of DNA conformational dynamics and interfacial hydration from direct readout via chemically defined deoxyguanosine derivatives. As a control, the substitution of G1 with 2-aminopurine (d2-AP) eliminates O6 in the major groove, resulting in low-affinity binding. Exchanging O6 with N2H2 in G1 (iso-dG/iso-d5mC) produced the same result, suggesting that C=O is an irreplaceable H-bond acceptor in ETS/DNA direct readout. Removal of NH2 (inosine) in the minor groove, opposite the protein-contact surface, impaired binding without altering the osmotic properties. Inosine substitution weakened local base-pairing, which suggests dynamics and hydration contributions in PU.1/DNA recognition could be separated. To explore this possibility, we assessed the binding of the high-affinity 5’-GIAA-3’ sequence to chimeric PU.1, where the DNA-contacting surfaces were partially interchanged with those from Ets-1, an ETS-family relative which is insensitive to osmotic pressure. The osmotically less sensitive chimeras were also less susceptible to inosine substitution. The modified nucleobases show that dynamic and hydration contributions (inosine, dI) may be separated from direct readout contributions (d2-AP, Iso-dG). The differential sensitivity of PU.1/Ets-1 chimeras to inosine-substituted DNA suggested an evolution of PU.1 from ETS relatives in which excess hydration is coupled to restraining DNA dynamics in complex formation.

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