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Effects of HU Binding on the Equilibrium Cyclization of Mismatched, Curved, and Normal DNA

Haribabu Arthanari ^*, Kristi Wojtuszewski , Ishita Mukerji  and Philip H. Bolton ^*

^* Chemistry Department and Molecular Biology and Biochemistry Department, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut

Correspondence: Address reprint requests to Ishita Mukerji, Tel.: 860-685-2422; Fax: 860-685-2141; E-mail: imukerji{at}wesleyan.edu or to Philip H. Bolton, Tel.: 860-685-2668; Fax: 860-685-2211; E-mail: pbolton{at}wesleyan.edu.

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 µM^-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.

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