Effects of HU binding on the equilibrium cyclization of mismatched, curved, and normal DNA.
The effects of HU, the histone-like protein from Escherichia coli, on the equilibrium cyclization of duplex DNAs have been observed as a function of protein concentration and DNA sequence. The results indicate that the presence of HU significantly enhances the extent of cyclization and increases the melting temperature, T(m), of the cyclized form of the DNA by >10 K. The stabilization of equilibrium cyclization by HU binding is at least -1.2 kcal/mol. The results are consistent with two HU homotypic dimers binding to each of the three 29-mer duplexes studied. One of the 29-mer duplexes contains a central dA tract, one contains mismatched sites, and one a conventional sequence. Stepwise or microscopic association constants, determined from the fluorescence data, range from 1.5 to 0.6 micro M(-1). The binding affinity of the HU dimer is strongest for the mismatched duplex and lowest for the dA tract, consistent with HU dimers having a preference for flexible DNA substrates. These results demonstrate the utility of the equilibrium cyclization approach to monitor DNA-protein interactions. These results have been considered along with those previously obtained to refine a model for the interaction of HU with duplex DNA.
Arthanari
Haribabu
H
Chemistry Department, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut, USA.
Wojtuszewski
Kristi
K
Mukerji
Ishita
I
Bolton
Philip H
PH
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D046911:Macromolecular Substances; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C116822:hns protein, E coli; D004247:DNA