Enzyme-Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer.

Abstract

Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) that are formed on DNA upon exposure of cells to ultraviolet light. The light-driven electron-transfer mechanism by which photolyase catalyzes the CPD monomerization after the enzyme-substrate complex has formed has been studied extensively. However, much less is understood about how photolyase recognizes CPDs on DNA. It has been clearly established that photolyase, like many other DNA-repair proteins, requires flipping of the CPD site into an extrahelical position. Photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base. In this paper, we perform direct measurements of photolyase binding to CPD-containing undecamer DNA that has been labeled with a fluorophore. We find that the association constant of ∼2 × 10(6) M(-1) is independent of the location of the CPD on the undecamer DNA. The binding kinetics of photolyase are best described by two rate constants. The slower rate constant is ∼10(4) M(-1) s(-1) and is most likely due to steric interference of the fluorophore during the binding process. The faster rate constant is on the order of 2.5 × 10(5) M(-1) s(-1) and reflects the binding of photolyase to the CPD on the DNA. This result indicates that photolyase finds and binds to a CPD lesion 100-4000 times slower than other DNA-repair proteins. In light of the existing literature, we propose a mechanism in which photolyase recognizes a CPD that is flipped into an extrahelical position via a three-dimensional search.