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Molecular and Cellular Biology, October 2009, p. 5465-5476, Vol. 29, No. 20
0270-7306/09/$08.00+0     doi:10.1128/MCB.00641-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression{triangledown}

Ellena V. McCarthy,1,{dagger} Julie E. Baggs,2 Jeanne M. Geskes,2 John B. Hogenesch,2 and Carla B. Green1*

Department of Biology, University of Virginia, Charlottesville, Virginia,1 Pharmacology Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania2

Received 18 May 2009/ Returned for modification 4 July 2009/ Accepted 9 August 2009

CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.

* Corresponding author. Present address: Department of Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., NB4.204G, Dallas, TX 75390-7433. Phone: (214) 648-7433. E-mail: carla.green{at}utsouthwestern.edu

{triangledown} Published ahead of print on 17 August 2009.

{dagger} Present address: Department of Cellular and Molecular Physiology, Yale School of Medicine, 333 Cedar St., New Haven, CT 06520.

Molecular and Cellular Biology, October 2009, p. 5465-5476, Vol. 29, No. 20
0270-7306/09/$08.00+0     doi:10.1128/MCB.00641-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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