Interaction of HMG proteins and H1 with hybrid PNA–DNA junctions

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‐PNA₁ and 4WJ‐PNA₃). 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‐PNA₃, like J1, shifts readily from an open to stacked conformation in the presence of Mg⁺², while 4WJ‐PNA₁ 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‐PNA₃ with nearly identical affinity (K_Ds) and 4WJ‐PNA₁ 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.