], were recently described (6, 7). Thus, it is evident that protein inheritance is a widespread phenomenon, at least in lower eukaryotes.The discovery of prions in yeast occurred in different ways. Some (i.e., [PSI + ] and [URE3]) were long known as genetic determinants of mysterious nature until their prion nature was proposed (8). The others were revealed by purposeful screening of potentially prionogenic proteins and corresponding determinants. The prion-like determinant [ISP + ], described in our earlier work (9), belongs to the first group, because it was detected as a nonchromosomal antisuppressor in strains containing specific sup35 nonsense suppressor mutations and the nonsense mutations his7-1 (UAA) and lys2-87 (UGA (Fig. 1A). The Sup + phenotype cosegregated with Ura + in tetrads of the diploid that was obtained by crossing the sfp1Δ and [ISP + ] strains (Fig. 1B). These findings indicate either that [ISP + ] is a prion form of Sfp1 or that the change in phenotype was caused by an independent manifestation of the SFP1-null allele.To distinguish between these two possibilities, the sfp1Δ strain was transformed with the centromeric vector pRS315-SFP1. Introduction of the wild-type SFP1 allele did not change the phenotype of the sfp1Δ strain [i.e., the absolute majority (556 of 559) of transformants has retained the Sup + phenotype]. This fact suggests that the change of phenotype in the sfp1Δ strain was caused by [ISP + ] loss rather than phenotypic effects of the SFP1 deletion; otherwise, restoration of the Sup − phenotype would be observed. Notably, this loss was irreversible, because we have not observed a single example of Sup -clones appearing in the mitotic progeny of sfp1Δ strains in contrast to [isp -] strains obtained by GuHCl treatment, which produced Sup -clones at a high frequency (9). These results confirmed that SFP1 could be considered as a likely host gene for [ISP + ].

SUMMARY Triplex structure-forming GAA/TTC repeats pose a dual threat to the eukaryotic genome integrity. Their potential to expand can lead to gene inactivation, the cause of Friedreich’s ataxia disease in humans. In model systems, long GAA/TTC tracts also act as chromosomal fragile sites that can trigger gross chromosomal rearrangements. The mechanisms that regulate the metabolism of GAA/TTC repeats are poorly understood. We have developed an experimental system in the yeast Saccharomyces cerevisiae that allows us to systematically identify genes crucial for maintaining the repeat stability. Two major groups of mutants defective in DNA replication or transcription initiation are found to be prone to fragility and large-scale expansions. We demonstrate that problems imposed by the repeats during DNA replication in actively dividing cells and during transcription initiation in nondividing cells can culminate in genome instability. We propose that similar mechanisms can mediate detrimental metabolism of GAA/TTC tracts in human cells.

Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved β-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds.

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3′ to 5′exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.

The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI+], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S. cerevisiae bearing a deleted or modified Sup35 N-terminal domain. In this work, we identified protein determinants of [NSI+] using an original method of proteomic screening for prions. The suppression of nonsense mutations in [NSI+] strains is determined by the interaction between [SWI+] and [PIN+] prions. Using genetic and biochemical methods, we showed that [SWI+] is the key determinant of this nonsense suppression, whereas [PIN+] does not cause nonsense suppression by itself but strongly enhances the effect of [SWI+]. We demonstrated that interaction of [SWI+] and [PIN+] causes inactivation of SUP45 gene that leads to nonsense suppression. Our data show that prion interactions may cause heritable traits in Saccharomyces cerevisiae.