Nucleic Acids Research Advance Access published online on September 4, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn551
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Molecular Biology |
Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA
1Department of Physics, Northeastern University, 111 Dana Research Center, 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, 3Department of Physics and Astronomy, University of Texas at Brownsville, 80 Fort Brown, Brownsville, TX 78520 4Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455 and 5Center for Interdisciplinary Research on Complex Systems, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA
*To whom correspondence should be addressed. Tel: +1 617 373 7323; Fax: +1 617 373 2943; Email: mark{at}neu.edu.
Correspondence may also be addressed to Ioulia Rouzina. Tel: +1 612-624-7468; Fax: +1 612-624-5121; Email: rouzi002{at}umn.edu
Received July 7, 2008. Revised August 6, 2008. Accepted August 12, 2008.
Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.
Present address: Boriana Marintcheva, Department of Biological Sciences, Bridgewater State College, Bridgewater, MA 02325, USA
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