Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono-and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.

The Neurospora clock protein FREQUENCY (FRQ) inhibits its transcriptional activator WHITE COLLAR COMPLEX (WCC) in a negative feedback loop and supports its accumulation in a positive loop. We show that positive feedback is a delayed effect of negative feedback underlying the same post-translational mechanisms: DNA-binding-competent active WCC commits rapidly to degradation. FRQ-dependent phosphorylation of WCC, which interferes with DNA binding (negative feedback), leads to reduced turnover and slow accumulation of newly expressed WCC (positive feedback). When DNA binding of WCC is compromised by mutation, its accumulation is independent of FRQ. Cycles of FRQ-dependent inactivation and PP2A-dependent reactivation of WCC occur in the minute range and are coupled to obligate rapid cycles of nucleo-cytoplasmic shuttling. WCC shuttling and activity cycles are modulated by FRQ in circadian fashion. We show here that FRQ supports negative and positive limbs of the clock by the same molecular mechanisms. Positive feedback (FRQ-dependent accumulation of WCC) is a delayed consequence of negative feedback (FRQ-dependent inactivation of WCC) rather than a mechanistically distinct feedback loop: WCC is active when FRQ is low or absent. Our data indicate that DNAbinding-competent, active WCC is unstable and rapidly turned over. FRQ-dependent phosphorylation of WCC interferes with DNA binding. This results in reduced turnover and allows accumulation of newly expressed WCC. Inactivation and reactivation of WCC are coupled to cycles of nucleo-cytoplasmic shuttling. We show that PP2A/RGB-1 activity is cytoplasmic, and hence passage of the WCC through the cytosol is obligatory for reactivation. Surprisingly, phosphorylation and shuttling cycles occur in the range of minutes and are modulated by FRQ in circadian fashion. Results and DiscussionWe investigated whether FRQ affects turnover of the WCC. In wild type, WCC is stable in constant darkness (DD) but turned over rapidly in constant light (LL) (Lee et al. 2000). To assess the influence of FRQ on WCC turnover, cultures of wild type and frq 9 , a mutant strain harboring a nonfunctional frq allele, were grown in LL. Turnover kinetics were then measured in the presence of cycloheximide (CHX). Degradation of WCC was substantially faster in frq 9 (t 1/2 ∼ 2.4 h) than in wild type (t 1/2 ∼ 4.2 h), demonstrating that FRQ stabilizes the light-activated WCC (Fig. 1A,F).In the negative feedback loop, FRQ promotes phosphorylation of WCC, which leads to its inactivation (Schafmeier et al. 2005). To investigate whether WCC activity affects its stability, we analyzed turnover of WCC in the wc-2G3 strain (Linden et al. 1997). wc-2G3 encodes a WC-2 version that lacks the C-terminal Zincfinger (Zn-finger) domain (Fig. 1B) and is henceforth referred to as wc2⌬C. WC-1 is unstable and does not accumulate in the absence of its assembly partner WC-2 (Cheng et al. 2002). WC-1 accumulated in high levels in wc-2⌬C (Fig. 1C), demonstrating that it assembled with the truncated WC-2⌬C. However, the mutan...

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Due to their health benefits there is much interest in developing microbial processes for efficient production of polyunsaturated fatty acids (PUFAs). In this study we co-expressed Mucor rouxii Δ(12) - and Δ(6) -desaturase genes in Saccharomyces cerevisiae, which resulted in a yeast strain that accumulated linoleic acid and γ-linolenic acid in the different lipid species. Additionally, the strain contained higher levels of phospholipids and lower levels of ergosterol than the reference strain. Integrated analysis of the transcriptome revealed decreased expression of genes involved in ergosterol biosynthesis, but more unexpectedly it also pointed towards attenuated activity of the ubiquitin-proteasome system and a reduced oxidative stress response. In vitro and in vivo measurements showed reduced levels of all three proteasomal activities and also increased levels of reactive oxidative species in the PUFA-producing strain. Overall our results clearly show that PUFAs in yeast can be detrimental for several key cellular pathways, such as the oxidative stress response and proteasomal activity, suggesting that the membrane composition is of vital importance for these processes.

FREQUENCY (FRQ) and the White Collar Complex (WCC), consisting of WC1 and WC2 subunits, are crucial components of positive and negative feedback loops of the circadian clock of Neurospora. In the positive limb, FRQ supports the accumulation of WC1 on a post-translational level and activates transcription of wc2. We analysed the transcriptional regulation of wc2. The WCC indirectly inhibits wc2 by controlling expression of a putative repressor. FRQ activates wc2 transcription by inhibiting WCC. A putative transcriptional activator binds to the wc2 promoter and antagonizes the repressor function. Furthermore, an internal promoter in the wc2 coding region drives expression of an amino-terminally shortened isoform, sWC2. Full-length WC2 and sWC2 are expressed in an antagonistic manner; thus, sWC2 expression seems to be a fail-safe mechanism that maintains total WC2 levels above a threshold.