The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

Iron-sulfur clusters (4Fe-4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol Î´) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol Î´ are the observations that Pol Î´ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.

GC content elevates mutation and recombination rates in the yeast Saccharomyces cerevisiae.

The chromosomes of many eukaryotes have regions of high GC content interspersed with regions of low GC content. In the yeast Saccharomyces cerevisiae, high-GC regions are often associated with high levels of meiotic recombination. In this study, we constructed URA3 genes that differ substantially in their base composition [URA3-AT (31% GC), URA3-WT (43% GC), and URA3-GC (63% GC)] but encode proteins with the same amino acid sequence. The strain with URA3-GC had an approximately sevenfold elevated rate of ura3 mutations compared with the strains with URA3-WT or URA3-AT About half of these mutations were single-base substitutions and were dependent on the error-prone DNA polymerase ζ. About 30% were deletions or duplications between short (5-10 base) direct repeats resulting from DNA polymerase slippage. The URA3-GC gene also had elevated rates of meiotic and mitotic recombination relative to the URA3-AT or URA3-WT genes. Thus, base composition has a substantial effect on the basic parameters of genome stability and evolution.

Feedback control of prion formation and propagation by the ribosome-associated chaperone complex.

Cross-beta fibrous protein aggregates (amyloids and amyloid-based prions) are found in mammals (including humans) and fungi (including yeast), and are associated with both diseases and heritable traits. The Hsp104/70/40 chaperone machinery controls propagation of yeast prions. The Hsp70 chaperones Ssa and Ssb show opposite effects on [PSI(+)], a prion form of the translation termination factor Sup35 (eRF3). Ssb is bound to translating ribosomes via ribosome-associated complex (RAC), composed of Hsp40-Zuo1 and Hsp70-Ssz1. Here we demonstrate that RAC disruption increases de novo prion formation in a manner similar to Ssb depletion, but interferes with prion propagation in a manner similar to Ssb overproduction. Release of Ssb into the cytosol in RAC-deficient cells antagonizes binding of Ssa to amyloids. Thus, propagation of an amyloid formed because of lack of ribosome-associated Ssb can be counteracted by cytosolic Ssb, generating a feedback regulatory circuit. Release of Ssb from ribosomes is also observed in wild-type cells during growth in poor synthetic medium. Ssb is, in a significant part, responsible for the prion destabilization in these conditions, underlining the physiological relevance of the Ssb-based regulatory circuit.

Dual role of ribosome-associated chaperones in prion formation and propagation.

Chaperones of the diverse ubiquitous Hsp70 family are involved in the regulation of ordered self-perpetuating protein aggregates (amyloids and prions), implicated in both devastating diseases and protein-based inheritance. Yeast ribosome-associated chaperone complex (RAC), composed of the Hsp40 protein Zuo1 and non-canonical Hsp70 protein Ssz1, mediates association of the Hsp70 chaperone Ssb with translating ribosomes. Ssb participates in co-translational protein folding, regulation of premature translation termination, and ribosome biogenesis. The loss of Ssb or disruption of RAC results in the increased formation of [PSI +], a prion form of the translation termination factor Sup35 (eRF3). This implicates co-translational protein misfolding in de novo prion formation. However, RAC disruption also destabilizes pre-existing [PSI +] prions, as Ssb, released from ribosomes to the cytosol in the absence of RAC, antagonizes the function of the major cytosolic chaperone, Ssa, in prion propagation. The mechanism of the Ssa/Ssb antagonism is currently under investigation and may include a competition for substrates and/or co-chaperones. Notably, yeast cells with wild-type RAC also release Ssb to the cytosol in certain unfavorable growth conditions, and Ssb contributes to increased prion loss in these conditions. This indicates that the circulation of Ssb between the ribosome and cytosol may serve as a physiological regulator of the formation and propagation of self-perpetuating protein aggregates. Indeed, RAC and Ssb modulate toxicity of some aggregating proteins in yeast. Mammalian cells lack the Ssb ortholog but contain a RAC counterpart, apparently recruiting other Hsp70 protein(s). Thus, amyloid modulation by ribosome-associated chaperones could be applicable beyond yeast.

Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock.

Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [PSI+ ], a prion isoform of the yeast translation termination factor Sup35 This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [PSI+ ] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [PSI+ ], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104-GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [PSI+ ], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone.

Yeast Short-Lived Actin-Associated Protein Forms a Metastable Prion in Response to Thermal Stress.

Self-perpetuating ordered protein aggregates (amyloids and prions) are associated with a variety of neurodegenerative disorders. Although environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. We have employed endogenous yeast prions as a model system to study environmental control of amyloid formation. A short-lived actin-associated yeast protein Lsb2 can trigger prion formation by other proteins in a mode regulated by the cytoskeleton and ubiquitin-dependent processes. Here, we show that such a heterologous prion induction is due to the ability of Lsb2 to form a transient prion state, generated in response to thermal stress. Evolutionary acquisition of prion-inducing activity by Lsb2 is traced to a single amino acid change, coinciding with the acquisition of thermotolerance in the Saccharomyces yeast lineage. This raises the intriguing possibility that the transient prion formation could aid in functioning of Lsb2 at higher temperatures.

Regulation of chaperone effects on a yeast prion by cochaperone Sgt2.

Yeast prions, based on self-seeded highly ordered fibrous aggregates (amyloids), serve as a model for human amyloid diseases. Propagation of yeast prions depends on the balance between chaperones of the Hsp100 and Hsp70 families. The yeast prion [PSI(+)] can be eliminated by an excess of the chaperone Hsp104. This effect is reversed by an excess of the chaperone Hsp70-Ssa. Here we show that the actions of Hsp104 and Ssa on [PSI(+)] are modulated by the small glutamine-rich tetratricopeptide cochaperone Sgt2. Sgt2 is conserved from yeast to humans, has previously been implicated in the guided entry of tail-anchored proteins (GET) trafficking pathway, and is known to interact with Hsps, cytosolic Get proteins, and tail-anchored proteins. We demonstrate that Sgt2 increases the ability of excess Ssa to counteract [PSI(+)] curing by excess Hsp104. Deletion of SGT2 also restores trafficking of a tail-anchored protein in cells with a disrupted GET pathway. One region of Sgt2 interacts both with the prion domain of Sup35 and with tail-anchored proteins. Sgt2 levels are increased in response to the presence of a prion when major Hsps are not induced. Our data implicate Sgt2 as an amyloid "sensor" and a regulator of chaperone targeting to different types of aggregation-prone proteins.