The Ccr4--not complex.

Hydroxyurea (HU) is mostly referred to as an inhibitor of ribonucleotide reductase (RNR) and as the agent that is commonly used to arrest cells in the S-phase of the cycle by inducing replication stress. It is a well-known and widely used drug, one which has proved to be effective in treating chronic myeloproliferative disorders and which is considered a staple agent in sickle anemia therapy and—recently—a promising factor in preventing cognitive decline in Alzheimer’s disease. The reversibility of HU-induced replication inhibition also makes it a common laboratory ingredient used to synchronize cell cycles. On the other hand, prolonged treatment or higher dosage of hydroxyurea causes cell death due to accumulation of DNA damage and oxidative stress. Hydroxyurea treatments are also still far from perfect and it has been suggested that it facilitates skin cancer progression. Also, recent studies have shown that hydroxyurea may affect a larger number of enzymes due to its less specific interaction mechanism, which may contribute to further as-yet unspecified factors affecting cell response. In this review, we examine the actual state of knowledge about hydroxyurea and the mechanisms behind its cytotoxic effects. The practical applications of the recent findings may prove to enhance the already existing use of the drug in new and promising ways.

/pdf/hydroxyurea-the-good-the-bad-and-the-ugly-51b794e1ip.pdf

Hydroxyurea-The Good, the Bad and the Ugly.

The ability to distinguish nonself from self is a fundamental characteristic of biological systems. In the filamentous fungus Neurospora crassa, multiple incompatibility genes mediate nonself recognition during vegetative growth. One of these genes, un-24, encodes both nonself recognition function and the large subunit of a type I ribonucleotide reductase, an evolutionarily conserved enzyme that is essential for the conversion of NDP precursors into dNDPs for use in DNA synthesis. Previous studies have shown that co-expression of the two allelic forms of un-24, Oakridge (OR) and Panama (PA), in the same cell results in cell death. We identify a 135 amino acid nonself recognition domain in the C-terminus region of UN-24 that confers an incompatibility-like phenotype when expressed in the yeast, Saccharomyces cerevisiae. Low-level expression of this domain results in several cytological and phenotypic characteristics consistent with an incompatibility reaction in filamentous fungi. These incompatibility phenotypes are correlated with the presence of a non-reducible complex consisting of the PA incompatibility domain and Rnr1p, a large subunit of ribonucleotide reductase in yeast. When the PA incompatibility domain is switched to high-level expression, the incompatibility phenotype transitions to wild-type concomitant with the appearance of a complex containing the PA incompatibility domain and Ssa1p, an Hsp70 homolog. Results from this study provide insights into the mechanism and control of vegetative nonself recognition mediated by ribonucleotide reductase in N. crassa, thus establishing the yeast system as a powerful tool to study fungal nonself recognition. Our work shows that heat shock proteins may function to deactivate vegetative incompatibility systems, as required for entry into the sexual cycle. Finally, our results suggest that variations on the PA incompatibility domain may serve as novel and specific antimicrobial peptides.

/pdf/trans-species-activity-of-a-nonself-recognition-domain-287cds4z9k.pdf

Trans-species activity of a nonself recognition domain

The mRNA deadenylation process, catalyzed by the CCR4 deadenylase, is known to be the major factor controlling mRNA decay rates in Saccharomyces cerevisiae. We have identified the proline-rich region and RRM1 domains of poly(A) binding protein (PAB1) as necessary for CCR4 deadenylation. Deletion of either of these regions but not other regions of PAB1 significantly reduced PAB1-PAB1 protein interactions, suggesting that PAB1 oligomerization is a required step for deadenylation. Moreover, defects in these two regions inhibited the formation of a novel, circular monomeric PAB1 species that forms in the absence of poly(A). Removal of the PAB1 RRM3 domain, which promoted PAB1 oligomerization and circularization, correspondingly accelerated CCR4 deadenylation. Circular PAB1 was unable to bind poly(A), and PAB1 multimers were severely deficient or unable to bind poly(A), implicating the PAB1 RNA binding surface as critical in making contacts that allow PAB1 self-association. These results support the model that the control of CCR4 deadenylation in vivo occurs in part through the removal of PAB1 from the poly(A) tail following its self-association into multimers and/or a circular species. Known alterations in the P domains of different PAB proteins and factors and conditions that affect PAB1 self-association would, therefore, be expected to be critical to controlling mRNA turnover in the cell.

PAB1 Self-Association Precludes Its Binding to Poly(A), Thereby Accelerating CCR4 Deadenylation In Vivo

The mammalian Ccr4-Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. In the nucleus, it is involved in the regulation of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, nuclear RNA surveillance, and DNA damage repair. In the cytoplasm, the Ccr4-Not complex plays a central role in mRNA decay and affects protein quality control. Most of our original knowledge of the Ccr4-Not complex is derived, primarily, from studies in yeast. More recent studies have shown that the mammalian complex has a comparable structure and similar properties. In this review, we summarize the evidence for the multiple roles of both the yeast and mammalian Ccr4-Not complexes, highlighting their similarities.

The Regulatory Properties of the Ccr4-Not Complex.

In Saccharomyces cerevisiae , DNA replication stress activates the replication checkpoint, which slows S-phase progression, stabilizes slowed or stalled replication forks, and relieves inhibition of the ribonucleotide reductase (RNR) complex. To identify novel genes that promote cellular viability after replication stress, the S. cerevisiae non-essential haploid gene deletion set (4812 strains) was screened for sensitivity to the RNR inhibitor hydroxyurea (HU). Strains bearing deletions in either CCR4 or CAF1 / POP2 , which encode components of the cytoplasmic mRNA deadenylase complex, were particularly sensitive to HU. We found that Ccr4 cooperated with the Dun1 branch of the replication checkpoint, such that ccr4 Δ dun1 Δ strains exhibited irreversible hypersensitivity to HU and persistent activation of Rad53. Moreover, because ccr4 Δ and chk1 Δ exhibited epistasis in several genetic contexts, we infer that Ccr4 and Chk1 act in the same pathway to overcome replication stress. A counterscreen for suppressors of ccr4 Δ HU sensitivity uncovered mutations in CRT1 , which encodes the transcriptional repressor of the DNA-damage-induced gene regulon. Whereas Dun1 is known to inhibit Crt1 repressor activity, we found that Ccr4 regulates CRT1 mRNA poly(A) tail length and may subtly influence Crt1 protein abundance. Simultaneous overexpression of RNR2 , RNR3 and RNR4 partially rescued the HU hypersensitivity of a ccr4 Δ dun1 Δ strain, consistent with the notion that the RNR genes are key targets of Crt1. These results implicate the coordinated regulation of Crt1 via Ccr4 and Dun1 as a crucial nodal point in the response to DNA replication stress.

/pdf/ccr4-contributes-to-tolerance-of-replication-stress-through-19ausl8het.pdf

Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length

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Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length