Interaction of HMG Proteins and H1 with Hybrid PNA-DNA Junctions

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Chemistry and Biochemistry


The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein-binding surface of an immobilized four-way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ-PNA(1) and 4WJ-PNA(3)). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two-state equilibrium between: (i) an open-x state favored by absence of Mg2+, low salt, and protein binding, and (ii) a compact stacked-x state favored by Mg2+. 4WJ-PNA(3), like J1, shifts readily from an open to stacked conformation in the presence of Mg+2, while 4WJ-PNA(1) does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ-PNA(3) with nearly identical affinity (K(D)s) and 4WJ-PNA(1) with two-fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.

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Protein Science





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