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Passage through mitosis is required to reset replication origins for the subsequent S phase. During mitosis, a series of biochemical reactions involving cyclin-dependent kinases (CDKs), the anaphase promoting complex or cyclosome (APC/C), and a mitotic exit network including Cdc5, 14, and 15 coordinates the proper separation and segregation of sister chromatids. Here we show that cyclin B/CDK inactivation can drive origin resetting in either early S phase or mitosis. This origin resetting occurs efficiently in the absence of APC/C function and mitotic exit network function. We conclude that CDK inactivation is the single essential event in mitosis required to allow pre-RC assembly for the next cell cycle.

Circadian rhythms are rhythms in gene expression, metabolism, physiology, and behavior that persist in constant environmental conditions with a cycle length near 24 h. In mammals, the circadian timing system is hierarchical. The primary pacemaker regulating circadian behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Most cell types express circadian clock genes and will express rhythmicity in vitro. In vivo, the SCN entrains peripheral oscillators through a complex set of physiological and hormonal rhythms (31, 32, 36). At the molecular level, circadian oscillations are governed by a cell-autonomous negative-feedback loop in which transcription factors drive the expression of their own negative regulators, leading to oscillation between periods of transcriptional activation and repression (reviewed in references 32 and 36). The bHLH-PAS containing transcription factors CLOCK or NPAS2 form heterodimers with BMAL1. These heterodimers binds to E-box elements within regulatory regions of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes to stimulate their transcription. Approximately 12 h after transcriptional activation, PER and CRY proteins reach concentrations sufficient to form repressor complexes that inhibit the activity of the CLOCK/NPAS2:BMAL1 heterodimer, reducing the transcription of Per and Cry genes and subsequently relieving PER/CRY-mediated negative feedback. E-box-mediated expression of other transcription factors, including members of the DBP/HLF/TEF and nuclear orphan receptor families (e.g., Rev-Erbα and ROR-A), provides a mechanism for clock control of genes with diverse promoters and with gene expression peaks occurring at a variety of phases. Posttranslational modifications of circadian clock proteins play a well-established role in the regulation of circadian cycle length. In both flies and mammals, phosphorylation of PER proteins by casein kinase 1 (CK1) proteins is thought to play a key step in determining the speed of the circadian clock (reviewed in reference 5). Mammalian cell culture studies indicate that the phosphorylation of PER proteins by CK1 epsilon (CK1ɛ) regulates their subcellular localization, likely affects their transcription repression capability, and promotes their degradation through a proteasomal pathway dependent upon the F-box proteins β-TrCP1 and β-TrCP2 (12, 29, 34, 35). Interference with β-TrCP1 activity lengthens circadian period in oscillating fibroblasts (27). The CK1 inhibitor IC261 and the proteasome inhibitors MG132 and lactacystin also lengthen period in fibroblasts (12). CRY proteins are also subjected to phosphorylation and degradation cycles that regulate circadian period. The F-box protein FBXL3 plays a key role in regulating CRY1 stability; mutations inactivating this gene increase circadian cycle length (6, 16, 30). Collectively, these studies indicate that the duration of activity of the PER/CRY repressor complex, regulated primarily by the stability of PER and CRY proteins, dictates the cycle length of the molecular oscillator (15). Genetic studies also support an important role for casein kinase action on PER proteins in regulating circadian period. A mutation in the Syrian hamster CK1ɛ gene, tau, shortens the circadian period of behavioral rhythms. Biochemically, the tau mutation (CK1ɛtau, a T178C substitution) differentially affects the activity of the kinase protein, reducing general kinase activity while increasing activity at specific residues of the PER proteins (14, 23). The tau mutation is a gain-of-function mutation with respect to circadian substrates, resulting in decreased PER stability and a reduction in circadian period length in tau mutant hamsters and mice (14, 24). In humans, familial advanced sleep phase syndrome (FASPS) is a circadian-based sleep disorder, in which affected individuals have a short circadian period and an advanced phase of the sleep-wake cycle. One study identified a FASPS pedigree with a mutation in human PER2 (hPER2; S662G mutation); this mutation prevents a priming phosphorylation, thus preventing CK1-mediated phosphorylation (33). A second study identified a dominant mutation within the kinase domain of CK1δ in a family with FASPS (38). Modeling this mutation in mice and flies revealed alterations in period length (38). In the circadian field, much of the attention on mammalian casein kinases has focused on CK1ɛ. The few studies examining CK1δ suggest that it plays a role similar to CK1ɛ. For example, CK1δ, like CK1ɛ, phosphorylates PER proteins, reducing their stability in vitro (7, 38), and both CK1δ and CK1ɛ are present in PER/CRY repressor complexes in vivo (21). Despite these similarities, the role of CK1δ in the molecular clockwork is not well understood. We report here results demonstrating important differences between CK1δ and CK1ɛ in the regulation of circadian cycle length, based on studies utilizing mice in which these genes have been inactivated.

Biological rhythms in mammals are driven by a central circadian clock located in the suprachiasmatic nucleus (SCN). At the molecular level the biological clock is based on the rhythmic expression of clock genes. Two basic helix-loop-helix (bHLH)/PAS-containing transcription factors, CLOCK and BMAL1 (MOP3), provide the basic drive to the system by activating transcription of negative regulators through E box enhancer elements. A critical feature of circadian timing is the ability of the clockwork to be entrained to the environmental light/dark cycle. The light-resetting mechanism of the mammalian circadian clock is poorly understood. Light-induced phase shifts are correlated with the induction of the clock genes mPer1 and mPer2 and a subsequent increase in mPER1 protein levels. It has previously been suggested that rapid degradation of BMAL1 protein in the rat SCN is part of the resetting mechanism of the central pacemaker. Our study shows that BMAL1 and CLOCK proteins are continuously expressed at high levels in the mouse SCN, supporting the hypothesis that rhythmic negative feedback plays the major role in rhythm generation in the mammalian pacemaker. Using both immunocytochemistry and immunoblot analysis, our studies demonstrate that BMAL1 protein in the mouse SCN is not affected by a phase-resetting light pulse. These results indicate that rapid degradation of BMAL1 protein is not a consistent feature of resetting mechanisms in rodents.

SummaryThe circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre-LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function.