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

• Published in: Protein science Vol. 22; no. 11; pp. 1552 - 1562
• Main Authors: Totsingan, Filbert, Bell, Anthony J.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.11.2013; Blackwell Publishing Ltd
• Subjects: Animals; C.D; Circular Dichroism; cruciform DNA; Deoxyribonucleic acid; DNA; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; four‐way junction; high mobility group protein B 1 (HMGB1); histone H1; Histones - chemistry; Histones - metabolism; HMGB1 Protein - chemistry; HMGB1 Protein - metabolism; Humans; Models, Molecular; Nucleic Acid Conformation; peptide nucleic acid (PNA); Peptide Nucleic Acids - chemistry; Peptide Nucleic Acids - metabolism; Protein Binding - physiology; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; four-way junction; histone H1; circular dichroism; peptide nucleic acid (PNA); high mobility group protein B 1 (HMGB1); cruciform DNA
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.2342

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 (KDs) 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.