Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair

VcCry1, a member of the CRY-DASH family, may serve two diverse roles <i>in vivo</i>, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling

Evidence from Thermodynamics that DNA Photolyase Recognizes a Solvent-Exposed CPD Lesion

Binding of a <i>cis</i>,<i>syn</i>-cyclobutane pyrimidine dimer (CPD) to <i>Escherichia coli</i> DNA photolyase was examined as a function of temperature, enzyme oxidation state, salt, and substrate conformation using isothermal titration calorimetry. While the overall Δ<i>G</i>° of binding was relatively insensitive to most of the conditions examined, the enthalpic and entropic terms that make up the free energy of binding are sensitive to the conditions of the experiment. Substrate binding to DNA photolyase is generally driven by a negative change in enthalpy. Electrostatic interactions and protonation are affected by the oxidation state of the required FAD cofactor and substrate conformation. The fully reduced enzyme appears to bind approximately two additional water molecules as part of substrate binding. More significantly, the experimental change in heat capacity strongly suggests that the CPD lesion must be flipped out of the intrahelical base stacking prior to binding to the protein; the DNA repair enzyme appears to recognize a solvent-exposed CPD as part of its damage recognition mechanism