Circadian clocks control many aspects of physiology and behavior and disruption of this timing system in mammals has been shown to result in significant health problems. In addition to causing major deleterious effects on metabolism (including obesity and diabetes), increased risk of some types of cancer and cardiovascular problems, recent evidence has also shown a close tie between circadian clocks and affective disorders, sleep abnormalities and major depression. Recent years have produced molecular models of how the circadian clock is built and how it maintains 24-hour periodicity. It is known that the core part of this clockwork is a negative feedback of transcription/translation whereby the clock proteins PERIOD (PER) 1, PER 2, CRYPTOCHROME (CRY) 1 and CRY 2 interact in a large repressive complex that represses their own synthesis. The rhythmic accumulation and decay of this repressive complex is thought to drive the rhythmic transcription of thousands of other genes that ultimately generate many rhythmic biochemical, neural and behavioral events. However, despite our basic understanding of this loop, there are still many unanswered questions. The specific roles of the various clock components are not well understood, the components of the CRY repressive complex are not fully characterized and the mechanism by which CRYs repress CLOCK/BMAL1 mediated transcription are not known. This proposal seeks to address these issues and test some key aspects of the circadian clock model through three specific aims. These aims will make use of a set of novel mutant alleles of the Cry1 and Cry2 genes. These alleles were identified in a random mutagenesis screen and have many different specific deficits in function. We will use these mutants as tools to probe the clock using a new cycling cell assay in which the mutant alleles will be expressed in a Cry null background. This approach will be complemented with biochemical analysis of the various components in these complexes. In the first Aim, we will examine the role of the PER proteins in the repressive activity and nuclear localization of the CRY-containing repressive complex. The second aim will analyze rhythmic CLOCK/BMAL1 binding and chromatin modifications of two cycling promoters in cells expressing different CRY mutants with various deficits in repression. This aim will also investigate some newly identified CRY-interacting proteins and examine their roles in CRY-mediated repression. In the third aim will use a panel of CRY chimeric mutants and proteomic analyses to investigate the functional differences between the two CRY proteins, which are structurally very similar but have non-redundant roles in the clock. These aims will test specific and important hypotheses about how the circadian clock in mammals actually works. Manipulation of the circadian mechanism is a potentially desirable treatment for many types of human disorders, but in order to design such treatment modalities, a more intricate understanding of the repressive arm of the circadian mechanism is critical.