title: Effects of pressure and temperature on the binding of RecA protein to single-stranded DNA.
abstract: The binding and polymerization of RecA protein to DNA is required for recombination, which is an essential function of life. We study the pressure and temperature dependence of RecA binding to single-stranded DNA in the presence of adenosine 5'-[γ-thio]triphosphate (ATP[γ-S]), in a temperature regulated high pressure cell using fluorescence anisotropy. Measurements were possible at temperatures between 5-60 °C and pressures up to 300 MPa. Experiments were performed on Escherichia coli RecA and RecA from a thermophilic bacteria, Thermus thermophilus. For E. coli RecA at a given temperature, binding is a monotonically decreasing and reversible function of pressure. At atmospheric pressure, E. coli RecA binding decreases monotonically up to 42 °C, where a sharp transition to the unbound state indicates irreversible heat inactivation. T. thermophilus showed no such transition within the temperature range of our apparatus. Furthermore, we find that binding occurs for a wider range of pressure and temperature for T. thermophilus compared to E. coli RecA, suggesting a correlation between thermophilicity and barophilicity. We use a two-state model of RecA binding/unbinding to extract the associated thermodynamic parameters. For E. coli, we find that the binding/unbinding phase boundary is hyperbolic. Our results of the binding of RecA from E. coli and T. thermophilus show adaptation to pressure and temperature at the single protein level.
authors: The Rockefeller University, Center for Studies in Physics and Biology, New York, NY 10021, USA.
authors: Jack
authors: J
authors: Merrin
authors: Pradeep
authors: P
authors: Kumar
authors: Albert
authors: A
authors: Libchaber
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001274:Atmospheric Pressure; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D011312:Pressure; D011485:Protein Binding; D055550:Protein Stability; D011938:Rec A Recombinases / Q000378:metabolism*; D013696:Temperature; D016963:Thermus thermophilus / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases