A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.     

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair

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Cold induction of EARLI1, a putative Arabidopsis lipid transfer protein, is light and calcium dependent

As sessile organisms, plants must adapt to their environment. One approach toward understanding this adaptation is to investigate environmental regulation of gene expression. Our focus is on the environmental regulation of EARLI1, which is activated by cold and long‐day photoperiods. Cold activation of EARLI1 in short‐day photoperiods is slow, requiring several hours at 4 °C to detect an increase in mRNA abundance. EARLI1 is not efficiently cold‐activated in etiolated seedlings, suggesting that photomorphogenesis is necessary for its cold activation. Cold activation of EARLI1 is inhibited in the presence of the calcium channel blocker lanthanum chloride or the calcium chelator EGTA. Addition of the calcium ionophore Bay K8644 results in cold‐independent activation of EARLI1. These data suggest that EARLI1 is not an immediate target of the cold response, and that calcium flux affects its expression. EARLI1 is a putative secreted protein and has motifs found in lipid transfer proteins. Over‐expression of EARLI1 in transgenic plants results in reduced electrolyte leakage during freezing damage, suggesting that EARLI1 may affect membrane or cell wall stability in response to low temperature stress.     

Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Recruitment of the homologous recombination machinery to sites of double‐strand breaks is a cell cycle‐regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B‐type cyclin/CDK1 activity. Induction of the intra‐S‐phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU‐treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra‐S‐phase checkpoint. We propose that B‐type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.     

Fluid replacement following dehydration reduces oxidative stress during recovery

To investigate the effects of hydration status on oxidative DNA damage and exercise performance, 10 subjects ran on a treadmill until exhaustion at 80% VO_2max during four different trials [control (C), 3% dehydration (D), 3% dehydration + water (W) or 3% dehydration + sports drink (S)]. Dehydration significantly decreased exercise time to exhaustion (D < C and S). Plasma MDA levels were significantly higher at pre-exercise in D than C. Plasma TAS was significantly lower at pre-exercise in C and S than in D, and was significantly lower in S than D at 60 min of recovery. Dehydration significantly increased oxidative DNA damage during exercise, but fluid replacement with water or sports drink alleviated it equally. These results suggest that (1) dehydration impairs exercise performance and increases DNA damage during exercise to exhaustion; and (2) fluid replacement prolongs exercise endurance and attenuates DNA damage.     

Intrauterine programming of ageing

Ageing is an unavoidable corollary to being alive; the most intuitive interpretation of ageing being that it is the consequence of progressive body degeneration. In agreement with this, current models propose that ageing occurs through a stepwise accumulation of DNA damage, which ultimately limits the regenerative capacity of tissues. On the other hand, there is increasing evidence that fetal distress can influence the development of disease in adult life, a phenomenon known as ‘intrauterine programming’. The extent to which an intrauterine exposure to DNA damage can compromise lifespan remains unclear. My group has recently generated a murine model of a human syndrome linked to defective DNA repair and observed that these animals age prematurely, but the accumulation of DNA damage is restricted mostly to the embryonic period. Here, I discuss the implications of this finding and propose that ageing can be influenced by fetal distress.     

The effect of starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius carassius)

Summary The effect of 16 weeks total starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius Carassius) has been investigated. In the white fibres the amount of myofibrillar material fell from 89.6% to 70.7% of the total fibre volume whilst in the red fibres the fall was from 72.2% to 70.3%. The sarcoplasmic reticulum appeared to have become swollen during starvation in both fibre types. In the white fibres the terminal cisternae of some triads seem to have fused. The volume of the red fibres occupied by mitochondria was reduced from 16.2 % to 5.9 %. The concentration of mitochondria in the white fibres was too low to detect any quantitative changes. A marked reduction in the amount of euchromatin material was observed in most white fibre nuclei and many red fibre nuclei. Many of the ultrastructural changes noted in the present study can be correlated with biochemical changes known to occur in the red and white myotomal muscles of fish during starvation. This work was supported by a grant from the Natural Environmental Research Council.     

Effect of arm-shoulder fatigue on carpenters at work

The purpose of the study was to analyse the effect of arm-shoulder fatigue on manual performance. Ten experienced carpenters performed three standardized tasks (nailing, sawing and screwing). Electromyographic activity was recorded from six arm-shoulder muscles and the performances were video-filmed. After 45 min of standardized arm-cranking (arm-shoulder-fatiguing exercise of approximately 70%-80% maximal oxygen consumption), the tasks were repeated. The number of work movements and the time taken for each task were recorded and the quality of the work performed was compared. After the fatiguing exercise, only nailing was perceived as being harder and more mistakes were made during nailing and sawing. Movement performance was not influenced during nailing but was slightly slower during sawing and faster during screwing. However, there were increased mean EMG amplitudes in the upper trapezius and biceps muscles during nailing, in the upper trapezius, anterior deltoid and infraspinatus muscles during sawing and in the anterior deltoid muscle during screwing. Of the muscles studied the upper trapezius and anterior deltoid muscles increased their activity most after the arm-shoulder-fatiguing exercise.     

Smooth muscle and myofibroblast-like cells in the perimedullary stromal basket of kidneys from ringed seals (Phoca hispida)

Summary The medullary pyramid of renculi in kidneys of ringed seals (Phoca hispida) is enclosed by a basket composed of ribbons of stromal tissue continuous with the wall of the calyx. Branched smooth muscle cells with well-developed Golgi complexes and rough endoplasmic reticulum and only an incomplete external lamina are the principal cells in sites near the origin of the ribbons from the calycal wall. Deeper in the corticomedullary junctional region, smooth muscle is progressively replaced with stellate or spindle-shaped cells exhibiting structural characteristics intermediate between those of fibroblasts and smooth muscle fibers. These myofibroblast-like cells contain arrays of parallel microfilaments 6–8 nm thick with associated focal densities and subplasmalemmal dense plaques, caveolae, elongate, often deeply wrinkled nuclei, and well-developed Golgi complexes and rough endoplasmic reticulum. Material resembling external lamina is associated with parts of the surfaces of most myofibroblast-like cells and intermediate junctions are present. Fibroblasts lacking arrays of parallel microfilaments are a minority at any level in the stromal ribbons. Interstitial cells in the vicinity of the corticomedullary junction show similar myofibroblast-like characteristics. The smooth muscle and myofibroblast-like cells presumably assist expression of urine from the papilla and calyx, and possibly participate as pacemakers for the urinary tract.