The Energetic Contribution of Induced Electrostatic Asymmetry to DNA Bending by a Site-Specific Protein

Abstract

DNA bending can be promoted by reducing the net negative electrostatic potential around phosphates on one face of the DNA, such that electrostatic repulsion among phosphates on the opposite face drives bending toward the less negative surface. To provide the first assessment of the energetic contribution to DNA bending when electrostatic asymmetry is induced by a site-specific DNA binding protein, we manipulated the electrostatics in the EcoRV endonuclease-DNA complex by mutation of cationic sidechains that contact DNA phosphates and/or by replacing a selected phosphate in each strand with uncharged methylphosphonate. Reducing the net negative charge at two symmetrically located phosphates on the concave DNA face contributes −2.3 to −0.9 kcal/mol (depending on position) to complex formation. In contrast, reducing negative charge on the opposing convex face produces a penalty of +1.3 kcal/mol. Förster resonance energy transfer experiments show that the extent of axial DNA bending (about 50°) is little affected in the modified complexes, implying that modification affects the energetic cost but not the extent of DNA bending. Kinetic studies show that favorable effects of induced electrostatic asymmetry on equilibrium binding derive primarily from a reduced rate of complex dissociation, suggesting stabilization of the specific complex between protein and markedly bent DNA. A smaller increase in the association rate may suggest that the DNA in the initial encounter complex is mildly bent. The data imply that protein-induced electrostatic asymmetry makes a significant contribution to DNA bending, but is not itself sufficient to drive full bending in the specific EcoRV-DNA complex.

Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation

“…2 that the amplitude of the jump in the SH signal increased as the concentration of Mg 2þ increased. Because the SH jump occurs before a significant portion of DNA is cleaved, the Mg 2þ acts at early times as an electrolyte that screens the repulsive interactions between the neighboring phosphate groups (3,(41)(42)(43). It thereby facilitates the binding of EcoR1 to DNA and allows for a more facile bending of DNA at the reaction site.…”

“…The total SH intensity, I 2ω ðtotalÞ is the incoherent sum of the contributions made by the individual particles to the SH intensity, where I 2 ωðjÞ is proportional to the absolute square of the second harmonic electric field generated by the microparticle j, i.e., I 2ω ðjÞ ∼ jE 2ω ðjÞj 2 . If the interface is charged, as it is in the experiments described here, largely due to the attachment of DNA molecules to the microparticle surface, then there is an additional source of SH light due to the third-order polarization, P ð3Þ 2ω , of the interfacial species, which is a product of three electric fields given by, P ð3Þ 2ω ¼ χ ð3Þ E ω E ω E static ðrÞ; [3] where E static ðrÞ is the electric field, predominantly due to DNA, at a distance, r, from the microparticle that polarizes the surrounding molecules, and χ ð3Þ is the third-order susceptibility. It is because E static ðrÞ is a zero frequency electric field that the third-order polarization generates light at 2ω.…”

“…The endonucleases, because of their capability to bind and cleave specific DNA sequences, also play a key role in DNA recombinant and cloning methods (1,2). The EcoR1-DNA equilibria and reaction dynamics have been probed extensively by a number of established methods, such as, but not limited to: FRET (3)(4)(5), gel electrophoresis (6)(7)(8)(9)(10)(11)(12)(13)(14)(15), surface plasmon resonance (16), microfluidic trapping (17), two-color cross-correlation spectroscopy (18), and isothermal calorimetry (19).…”

ESR spectroscopy identifies inhibitory Cu 2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

“…Experiments assessing Cu 2þ enhancement of binding used the same double-strand deoxyoligonucleotide TCGCGAATTCGCG as that in the ESR experiments. Equilibrium association constants (K A ) for wild-type EcoRI and His114Tyr (AECu 2þ ) were measured by the nitrocellulose filter binding assay as described (34,55). For assays in the absence of Cu 2þ , EDTA (1 mM) was included in the binding buffer (30 mM NEM, 10% glycerol, 10% dioxane, 100 μM dithiothreitol, 100 μg∕mL bovine serum albumin) plus 0.3 M KCl; pH 8.0, 22°C.…”

“…4A). Divalent metals generally promote protein-DNA binding by decreasing the charge repulsion between the protein and nearby DNA phosphates (15,33,34). For example, we previously showed that the competitive inhibitor Ca 2þ binds to EcoRI at the negative charge cluster in the active site and thereby increases the equilibrium association constant (K A ) as much as 380-fold (15).…”