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.