The circadian clock ensures successful DNA replication in cyanobacteria

Disruption of circadian rhythms causes decreased health and fitness, and evidence from multiple organisms links clock disruption to dysregulation of the cell cycle. However, the function of circadian regulation for the essential process of DNA replication remains elusive. Here, we demonstrate that in the cyanobacterium Synechococcus elongatus, a model organism with the simplest known circadian oscillator, the clock generates rhythms in DNA replication to minimize the number of open replication forks near dusk that would have to complete after sunset. Metabolic rhythms generated by the clock ensure that resources are available early at night to support any remaining replication forks. Combining mathematical modeling and experiments, we show that metabolic defects caused by clock–environment misalignment result in premature replisome disassembly and replicative abortion in the dark, leaving cells with incomplete chromosomes that persist through the night. Our study thus demonstrates that a major function of this ancient clock in cyanobacteria is to ensure successful completion of genome replication in a cycling environment.

Circadian clocks, internally generated rhythms in physiology with a ∼24 h period, are found in all domains of life. These clocks allow organisms to coordinate their physiological activities in anticipation of the daily cycle in the external environmental (1–3). Disruption of clocks caused either by mutation or clock–environment mismatch leads to decreased health and reproductive fitness in multiple organisms (4–6). In mammals, risk for age-related diseases such as cancer and cardiometabolic dysfunction is enhanced by circadian disruption (7, 8).Although much is now understood about the molecular mechanisms that generate rhythms, the origin of these health defects is still incompletely understood. A common target of circadian control shared across many species is the progression of the cell cycle (9–12). In animals, disrupted circadian rhythms are often linked to aberrant cell proliferation and tumorigenesis (13). Successful duplication of the genome is essential for the production of viable progeny. Replicating a bacterial genome can take up to several hours, a timescale over which external illumination from sunlight can change substantially. We therefore speculated that initiation of DNA replication could be a key point of circadian control. The cyanobacterium Synechococcus elongatus, which has the simplest known circadian system, is a powerful model system to address these issues, both because its clock is intimately coupled to cell cycle (9, 14–16) and because clock–environment misalignment has profound effects on reproductive fitness (17). Here, we analyze whether replication is clock-regulated in S. elongatus and the consequences of clock–environment mismatch on DNA replication.     

Daily oscillations in liver function: diurnal vs circadian rhythmicity.

The rodent suprachiasmatic nucleus (SCN), a site in the brain that contains a light-entrained biological (circadian) clock, has been thought of as the master oscillator, regulating processes as diverse as cell division, reproductive cycles, sleep, and feeding. However, a second circadian system exists that can be entrained by meal feeding and has an influence over metabolism and behavior. Recent advances in the molecular genetics of circadian clocks are revealing clock characteristics such as rhythmic clock gene expression in a variety of non-neural tissues such as liver. Although little is known regarding the function of these clock genes in the liver, there is a large literature that addresses the capabilities of this organ to keep time. This time-keeping capability may be an adaptive function allowing for the prediction of mealtime and therefore improved digestion and energy usage. Consequently, an understanding of these rhythms is of great importance. This review summarizes the results of studies on diurnal and circadian rhythmicity in the rodent liver. We hope to lend support to the hypothesis that there are functionally important circadian clocks outside of the brain that are not light- or SCN-dependent. Rather, these clocks are largely responsive to stimuli involved in nutrient intake. The interaction between these two systems may be very important for the ability of organisms to synchronize their internal physiology.     

Intermolecular associations determine the dynamics of the circadian KaiABC oscillator

Three proteins from cyanobacteria (KaiA, KaiB, and KaiC) can reconstitute circadian oscillations in vitro. At least three molecular properties oscillate during this reaction, namely rhythmic phosphorylation of KaiC, ATP hydrolytic activity of KaiC, and assembly/disassembly of intermolecular complexes among KaiA, KaiB, and KaiC. We found that the intermolecular associations determine key dynamic properties of this in vitro oscillator. For example, mutations within KaiB that alter the rates of binding of KaiB to KaiC also predictably modulate the period of the oscillator. Moreover, we show that KaiA can bind stably to complexes of KaiB and hyperphosphorylated KaiC. Modeling simulations indicate that the function of this binding of KaiA to the KaiB•KaiC complex is to inactivate KaiA''s activity, thereby promoting the dephosphorylation phase of the reaction. Therefore, we report here dynamics of interaction of KaiA and KaiB with KaiC that determine the period and amplitude of this in vitro oscillator.     

Phototransduction cascade and circadian oscillator in chicken pineal gland

ABSTRACT: The chicken pineal gland has an endogenous circadian oscillator that controls the diurnal oscillation of N-acetyltransferase activity responsible for melatonin rhythm. It has been speculated that the chicken pineal cell contains a photoreceptive molecule that receives the environmental light signal and transmits the signal to the oscillator for resetting the phase. In spite of several lines of evidence suggesting the similarity between retinal and pineal photon-signal transducing proteins, the identity of the photoreceptive molecule had been an open question. In 1994, we isolated a pineal cDNA encoding a novel photoreceptive molecule and named it "pinopsin." The protein expressed in 293EBNA cells bound 11- cis -retinal to form a blue-sensitive pigment with an absorption maximum at about 470 nm. A putative G-protein interaction site of pinopsin shared a relatively high similarity in amino acid sequence to that of rhodopsin, implying that pinopsin functionally couples with transducin or transducin-like G-protein(s) in the pineal cells. We have cloned a cDNA for chicken pineal transducin α-subunit, and the deduced amino acid sequence contained a potential site to be ADP-ribosylated by pertussis toxin (PTX). Therefore, the transducin-mediated pathway could be blocked by PTX, though previous studies showed that treatment of the cultured chicken pineal cells with PTX had no effect on the light-induced phase-shift of the oscillator. Accordingly, it is unlikely that transducin mediates the light-input pathway to the oscillator, which may involve PTX-insensitive G-protein(s) or some unidentified component(s). The G-protein coupled receptor-mediated signaling processes regulating melatonin synthesis are discussed.     

昼夜节律变化对动脉粥样硬化形成的影响及其相关通路的研究进展

地球上几乎所有的生命都存在昼夜节律,控制昼夜节律的生物钟具有重要的生理功能,同时也是疾病的重要调节器.昼夜节律与动脉粥样硬化关系密切.研究表明受损的生物钟会影响造血过程和糖脂代谢,并改变局部斑块病变中的细胞功能.在分子水平上,昼夜节律可以通过Toll样受体(TLR)通路调节动脉粥样硬化炎症状态和血管重塑,通过蛋白激酶B...     

Circadian control of carbohydrate availability for growth in Arabidopsis plants at night

Plant growth is driven by photosynthetic carbon fixation during the day. Some photosynthate is accumulated, often as starch, to support nocturnal metabolism and growth at night. The rate of starch degradation in Arabidopsis leaves at night is essentially linear, and is such that almost all of the starch is used by dawn. We have investigated the timer that matches starch utilization to the duration of the night. The rate of degradation adjusted immediately and appropriately to an unexpected early onset of night. Starch was still degraded in an appropriate manner when the preceding light period was interrupted by a period of darkness. However, when Arabidopsis was grown in abnormal day lengths (28 h or 17 h) starch was exhausted ∼24 h after the last dawn, irrespective of the actual dawn. A mutant lacking the LHY and CCA1 clock components exhausted its starch at the dawn anticipated by its fast-running circadian clock, rather than the actual dawn. Reduced growth of wild-type plants in 28-h days and lhy/cca1 mutants in 24-h days was attributable to the inappropriate rate of starch degradation and the consequent carbon starvation at the end of night. Thus, starch degradation is under circadian control to ensure that carbohydrate availability is maintained until the next anticipated dawn, and this control is necessary for maintaining plant productivity.     

Sleep,circadian rhythms,and type 2 diabetes mellitus

Over the last 60 years we have seen a significant rise in metabolic disease, especially type 2 diabetes. In the same period, the emergence of electricity and artificial lighting has allowed our behavioural cycles to be independent of external patterns of sunlight. This has led to a corresponding increase in sleep deprivation, estimated to be about 1 hour per night, as well as circadian misalignment (living against the clock). Evidence from experimental animals as well as controlled human subjects have shown that sleep deprivation and circadian misalignment can both directly drive metabolic dysfunction, causing diabetes. However, the precise mechanism by which these processes contribute to insulin resistance remains poorly understood. In this article, we will review the new literature in the field and propose a model attempting to reconcile the experimental observations made. We believe our model will serve as a useful point of reference to understand how metabolic dysfunction can emerge from sleep or circadian rhythm disruptions, providing new directions for research and therapy.     

From the Cover: Feature Article: Light-arousal and circadian photoreception circuits intersect at the large PDF cells of the Drosophila brain

The neural circuits that regulate sleep and arousal as well as their integration with circadian circuits remain unclear, especially in Drosophila. This issue intersects with that of photoreception, because light is both an arousal signal in diurnal animals and an entraining signal for the circadian clock. To identify neurons and circuits relevant to light-mediated arousal as well as circadian phase-shifting, we developed genetic techniques that link behavior to single cell-type resolution within the Drosophila central brain. We focused on the unknown function of the 10 PDF-containing large ventral lateral neurons (l-LNvs) of the Drosophila circadian brain network and show here that these cells function in light-dependent arousal. They also are important for phase shifting in the late-night (dawn), indicating that the circadian photoresponse is a network property and therefore non-cell-autonomous. The data further indicate that the circuits underlying light-induced arousal and circadian photoentrainment intersect at the l-LNvs and then segregate.     

Bright daytime light enhances circadian amplitude in a diurnal mammal

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light–dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronization with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured circadian rhythms in behavioral activity, body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light–dark cycles at four different daytime intensities (ranging from 18 to 1,900 lx melanopic equivalent daylight illuminance). R. pumilio showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarization were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms and highlight the potential importance of daytime light exposure for circadian health.

In mammals, near-24-h (circadian) rhythms in physiology and behavior are orchestrated by a master clock located in the hypothalamic suprachiasmatic nuclei (SCN) (1, 2). The SCN clock generates a circadian rhythm in electrical activity, with neurons significantly more excited during the day (up state) than at night (down state) (3, 4). This endogenous rhythm is synchronized (entrained) to the external 24-h light–dark (LD) cycle via input from the retina (5). Thus, light exposure in the circadian night induces adjustments in circadian phase to ensure that internal time faithfully reflects external (celestial) time. Conceptual and mathematical models of light’s impact on the clock address this ability to reset circadian phase (the basis of entrainment) (6–9). However, there is evidence that another fundamental property of circadian rhythms, their amplitude, may also be influenced by light.It is well established that the amplitude and reliability of 24-h rhythms in some aspects of physiology and behavior can be enhanced by increasing daytime light exposure (10–20). Such effects may be explained by the ability of light to directly engage some of the systems under circadian control (e.g., increasing alertness and body temperature, Tb) (21–23) and thus enhance rhythm amplitude de facto, without impacting the circadian clock itself. However, reports that enhanced daytime light can also lead to higher production of melatonin on the subsequent night, many hours after light exposure has ceased (12, 24–26), pose a challenge to that explanation. The long-lasting nature of that effect raises the possibility that daytime light may have a more fundamental impact on circadian amplitude. We set out here to address this possibility by using laboratory rodents to ask whether increasing daytime irradiance produces persistent improvements in circadian amplitude and whether this can be traced back to changes in the physiological activity of the SCN circadian oscillator itself.A challenge to studying the impact of daytime light exposure in common laboratory models (mice, hamsters, and rats) is that they are nocturnal and employ strategies to avoid light in the day (such as curling up asleep). We therefore used a diurnal rodent, Rhabdomys pumilio (the four-striped mouse) (27–29) which is active through the day in both the laboratory and wild, ensuring good exposure to modulations in daytime light intensity. We find that increasing irradiance across a range equivalent to that from dim indoor lighting to natural daylight enhances the reproducibility and robustness of behavioral and physiological rhythms at the whole-animal level that persist into subsequent free run in constant darkness. This effect is associated with profound differences in the electrophysiological activity of the SCN, with bright daytime light producing persistent increases in SCN excitability and enhancing the amplitude of the circadian variation in spontaneous neuronal activity.     

Physiological consequences of space flight,including abnormal bone metabolism,space radiation injury,and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light–dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.     

A novel enzyme‐dependent melatonin metabolite in humans

Exogenous melatonin is widely used in humans for multiple pharmacologic purposes. The metabolic pathways of melatonin reflect the fate and functions of melatonin in vivo. This study was designed to re‐profile melatonin metabolism in humans using a metabolomic approach. In the urine of healthy subjects treated with 10 mg melatonin, sulfate‐ or glucuronide‐conjugated metabolites of melatonin were detected, including 6‐hydroxymelatonin sulfate, 6‐hydroxymelatonin glucuronide, N‐acetylserotonin glucuronide, N‐acetylserotonin sulfate, and an unknown sulfated metabolite (X). The molecular weight of metabolite X was 14 Da smaller than 6‐hydroxymelatonin sulfate, but 16 Da larger than N‐acetylserotonin sulfate. Further studies suggest that metabolite X was produced via O‐demethylation, 6‐hydroxylation, and sulfation. The antioxidant products of melatonin, N(1)‐acetyl‐N(2)‐formyl‐5‐methoxykynuramine and N(1)‐acetyl‐5‐methoxy‐kynuramine, were not detected in human urine. In summary, this study provided a global view of melatonin metabolism in humans and extended our knowledge of enzyme‐dependent pathways of melatonin metabolism.