Ubiquitylation of the 9-1-1 checkpoint clamp is independent of rad6-rad18 and DNA damage.

A recent report proposed a function of the ubiquitin conjugation factors Rad6 and Rad18 comparable to the bacterial SOS response, controlling damage-induced transcriptional activation and contributing to checkpoint signaling. The relevant ubiquitylation target was identified as budding yeast Rad17, a subunit of the PCNA-like 9-1-1 checkpoint clamp. We report here that in fact all three subunits of the 9-1-1 complex are ubiquitylated. However, in contrast to previous results, we found modification of Rad17 to be independent of DNA damage, the Rad6-Rad18 complex, the putative acceptor site (lysine 197), and loading of the complex onto DNA. Consistently, we were unable to observe enhanced damage sensitivity or defects in checkpoint signaling in a rad17(K197R) mutant. Instead, our findings suggest that ubiquitylation of the 9-1-1 complex may be a background reaction that in some cases can mediate proteasomal degradation.

Transcriptional regulation and function of the Neurospora clock gene white collar 2 and its isoforms.

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.

Postreplication repair and PCNA modification in Schizosaccharomyces pombe.

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.

Heterologous production of polyunsaturated fatty acids in Saccharomyces cerevisiae causes a global transcriptional response resulting in reduced proteasomal activity and increased oxidative stress.

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.

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo-cytoplasmic shuttling.

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.