Ahmad1,7* Abstract Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability Jacqueline Vieira1, Alex R. Jones2, Antoine Danon 1 , Michiyo Sakuma 2 , Nathalie Hoang 1 , David Robles1, Shirley Tait2, Derren J. Heyes2, Marie Picot3, Taishi Yoshii4, Charlotte Helfrich-Fo¨ rster5, Guillaume Soubigou6, Jean-Yves Coppee6, Andre´ Klarsfeld3, Francois Rouyer3, Nigel S. Scrutton 2 , Margaret 1 Universite´ Paris VI, Paris, France, 2 Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom, 3 Institut de Neurobiologie Alfred Fessard, CNRS UPR 2216 (NGI), Gif-sur-Yvette, France, 4 Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan, 5 University of Wu¨ rzburg, Biocentre, Neurobiology and Genetics, Wu¨ rzburg, Germany, 6 Institut Pasteur, Transcriptome and Epigenome Platform, Genomes and Genetics Department, Paris, France, 7 Penn State University, Media, Pennsylvania, United States of America Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism. Citation: Vieira J, Jones AR, Danon A, Sakuma M, Hoang N, et al. (2012) Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability. PLoS ONE 7(3): e31867. doi:10.1371/journal.pone.0031867 Editor: Alfredo Herrera-Estrella, Cinvestav, Mexico Received October 3, 2011; Accepted January 13, 2012; Published March 12, 2012 Copyright: 2012 Vieira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding is acknowledged from the Le Centre national de la recherche scientifique, ACI/BCMS and National Science Foundation (#0848311) to MA, from the Electromagnetic Fields Biological Research Trust (EMF BRT) to NS, and from the German Research Foundation (GRK 640) and the European Union (6th framework Project EUCLOCK, no. 018741) to CH, NH is recipient of an ACI/BCMS fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: margaret.ahmad@upmc.fr Introduction Cryptochromes are flavoprotein receptors characterized by striking sequence and structural homology to photolyases, a class of light sensing DNA repair enzyme. Cryptochromes retain the light sensing N-terminal domain of photolyases, but have largely lost DNA repair activity and instead developed novel roles in signaling [1,2]. Like photolyases, cryptochromes can bind both folate and flavin chromophores and undergo photochemical modification in response to light. In animals, there are two principal families of cryptochromes that have known roles in signaling, namely the Type I and the Type II cryptochromes, occurring in both vertebrates and invertebrates. These have significant homology to the 6-4 photoproduct repair class of photolyases and have roles in both the invertebrate and vertebrate circadian clock [3,4]. The N-terminal (photolyase-like) domains are highly conserved across both Type I and Type II cryptochromes, and the photolyases, and are thought to be involved in substrate recognition as well as light sensing, while the C-terminal region mainly modulates accessibility of the N-terminal domain to partner molecules [5,6]. Although much is known of the signaling pathways and interaction partners of both Type I and Type II cryptochromes and of their role in the different animal circadian clocks, the question of the light responsivity of animal cryptochromes, and the mechanism of converting the light signal into a biological response remains to be resolved. This is particularly the case for the Type II cryptochromes found in mammalian systems, including man [7]. It is currently known that animal type I cryptochromes, as exemplified by Drosophila cryptochrome (DmCRY), show robust light-dependent responses both in whole living flies and in cell culture systems [1]. DmCRY responses are observed in the blue/ near-UV region of the visible spectrum (below 500 nm) with sharp cut-off above 500 nm and a peak at 450 nm, which corresponds closely with the absorption spectrum of oxidized flavin in the visible spectrum [8,9]. Purified isolated Dmcry proteins are bound PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e31867