title: Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA.
abstract: 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.
authors.affiliation: Department of Physics, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA.
authors.forename: Leila
authors.initials: L
authors.lastname: Shokri
authors.forename: Boriana
authors.initials: B
authors.lastname: Marintcheva
authors.forename: Mootaz
authors.initials: M
authors.lastname: Eldib
authors.forename: Andreas
authors.initials: A
authors.lastname: Hanke
authors.forename: Ioulia
authors.initials: I
authors.lastname: Rouzina
authors.forename: Mark C
authors.initials: MC
authors.lastname: Williams
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000737:chemistry; D013816:Thermodynamics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; C060035:gp32 protein, Enterobacteria phage T4; D012965:Sodium Chloride; D004247:DNA
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