Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 133, Issue 7, Pages 2183-2191Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja107691w
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Funding
- Ministry of Education, Science, Sports and Culture in Japan [18205002, 20107003]
- National Institutes of Health [GM37684]
- International Center for Integrated Research and Advanced Education in Material Science, Kyoto University, Japan
- Skaggs Institute for Chemical Biology
- Grants-in-Aid for Scientific Research [20107003, 20107001] Funding Source: KAKEN
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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 h 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 mu s 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.
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