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.

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

“…Therefore, it appears that PL binds to substrate significantly slower by two to four orders of magnitude compared to what is reported for other site‐specific and sequence‐specific DNA proteins . In agreement with a proposal by Sancar and coworkers , the new evidence from our studies, that is the smaller substrate binding rate constant of 2.5 × 10 5 M −1 s −1 , the change in heat capacity upon substrate binding and the small contribution of electrostatic interactions to Δ G of substrate binding, all points to PL not using facilitated diffusion to find and bind to CPD lesions. Instead, the evidence from our and Sancar's work strongly indicate that EcPL performs a three‐dimensional diffusion‐controlled search for extrahelical, solvent‐exposed CPD lesions with only limited contributions from facilitated diffusion.…”

“…This hypothesis is further supported by direct measurement of PL binding to CPD as monitored by a fluorescent label on the DNA molecule . The second‐order rate constant for binding at 15°C was found to be 2.5 × 10 5 M −1 s −1 .…”

“…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.…”

Formation and Recognition of UV-Induced DNA Damage within Genome Complexity

“…Localization of the pyrimidine dimer in the enzymatic active binding pocket depends on a helical out-flipping of the damaged nucleotides ( Figure 3 a) [ 59 , 60 , 61 , 62 ]. The most recent study of CPD photolyase substrate binding kinetics suggests that conversely to other DNA repair proteins, CPD recognition does not rely on one-dimensional sliding or hopping along the DNA, but on the three-dimensional search for an extrahelical out-flipped photolesion [ 63 , 64 ]. Once out-flipped, the intrahelical bubble is stabilized by a bubble-intruding region (BIR) in Class II photolyases [ 65 , 66 ] and by a conserved Arg421 in 6-4PP photolyases ( Figure 3 a) [ 61 , 62 ].…”