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

Abstract

Binding of a cis,syn-cyclobutane pyrimidine dimer (CPD) to Escherichia coli DNA photolyase was examined as a function of temperature, enzyme oxidation state, salt, and substrate conformation using isothermal titration calorimetry. While the overall ΔG° 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.

A Review of Spectroscopic and Biophysical‐Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA

“…They also stated that the enzyme uses these α‐helices to flip the CPD out of the DNA duplex into the cavity. However, evidence from PL–substrate binding studies do not support an active role of the enzyme in CPD flipping ( vide infra ) . In a follow‐up study, they also found evidence of hydrogen bonding to a C4=O of the CPD and argued that the 5′‐C4 = O of the CPD is most likely forming a hydrogen bond with Glu275 (E283 in AnPL) .…”

“…build upon this foundation to report the first binding constant for purified protein (K A = 4.7–6 × 10 7 M −1 at 100 m m NaCl at 22°C) using a UV‐damaged pBR322 plasmid with a nitrocellulose filter assay. There are additional measurements of binding constants using different assay methods including gel retardation , surface plasmon resonance (SPR) , isothermal titration calorimetry (ITC) and fluorescence with each study using different substrates, temperatures and solvent conditions.…”

“… reported TtPL binding to a single‐stranded 7‐mer with a central CPD using SPR at 25°C; we presume the study used the FADH • state of the protein, the form of TtPL as isolated. Lastly, in 2011, Wilson, Crystal, Rohrbaugh and coworkers measured binding of both UV‐damaged p(dT) 10 and the dsDNA substrate to EcPL using ITC at 25°C; binding measurements were completed on all three oxidation states of EcPL . The data from the three studies were fit to the relationship shown in Eq.…”

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

“…The values obtained for EcPL with the same substrate but at pH 7.00 were different but also dependent on oxidation state: ΔH°values ranged from −27 kJ/mol for the PL FAD state to −40 kJ/mol for the PL FADH − state. 15 To summarize the experimental data, the electrochemistry and pH-dependent behavior of VcCRY1, a single-strand CPD photolyase and CRY-DASH protein, are significantly different from those of EcPL, the best understood CPD photolyase. Formation of the FAD state is blocked in VcCRY1 when UVdamaged DNA is bound.…”

Base Pair Opening in a Deoxynucleotide Duplex Containing a cis-syn Thymine Cyclobutane Dimer Lesion

“…Recent computational studies support the idea that the barrier to base flipping at the lesion site might be low, 16 and thermodynamic studies suggest that photolyase might bind an already extruded lesion. 17 …”