Light-Induced Conformational Change and Product Release in DNA Repair by (6−4) Photolyase

Abstract

Proteins of the cryptochrome/photolyase family share high sequence similarities, common folds and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24-hour circadian clock, whereas (6-4) photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/photolyase family proteins. The entire light-dependent DNA repair process for (6-4) photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The (6-4) photolyase by itself showed significant volume changes after blue light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 μs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair photolyases to access the entire genome in cells.

Eukaryotic Class II Cyclobutane Pyrimidine Dimer Photolyase Structure Reveals Basis for Improved Ultraviolet Tolerance in Plants

“…Thus, positioning a phosphate ion near the substrate and cofactor binding sites may provide both eukaryotic PHR families with control mechanisms for constraining and regulating their active sites. Phosphate-induced protein conformational changes could adjust intra-or intermolecular hydrogen bonds to aid electron transfer, DNA distortion, base flipping, or product release during the repair process (44).…”

Repair of the (6–4) Photoproduct by DNA Photolyase Requires Two Photons

“…where η 1 and η 2 are the quantum yields of the first and second photoreaction, respectively. Substrate binding and product release are expected15 to be much faster than the overall repair rate (less than 0.01 s −1 in all our experiments) and are hence considered to be non‐limiting (Supporting Information, Section 4).…”

DNA Repair by Reversal of DNA Damage

“…The investigators observed a key cyclic proton transfer step between an active-site histidine residue and the substrate, which occurs in 425 ps and leads to 6 -4 PP repair in tens of nanoseconds . In addition, time-resolved experiments of Xenopus laevis (624) photolyase uncovered a drastic diffusion change, which was assigned to the rapid dissociation (time constant of 50 ms) of the protein from the repaired DNA product (Kondoh et al 2011). Later on, 6-4 PP repair mediated by the same enzyme was investigated using Fourier transform infrared (FTIR) spectroscopy .…”