In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.

Mismatch repair (MMR) is a major DNA repair pathway in cells from all branches of life that removes replication errors in a strandspecific manner, such that mismatched nucleotides are preferentially removed from the newly replicated strand of DNA. Here we demonstrate a role for MMR in helping create new phenotypes in nondividing cells. We show that mispairs in yeast that escape MMR during replication can later be subject to MMR activity in a replication strand-independent manner in nondividing cells, resulting in either fully wild-type or mutant DNA sequence. In one case, this activity is responsible for what appears to be adaptive mutation. This replication strand-independent MMR activity could contribute to the formation of tumors arising in nondividing cells and could also contribute to mutagenesis observed during somatic hypermutation of Ig genes.DNA mismatch repair | oligonucleotide transformation | 8-oxoG D NA mismatch repair (MMR) recognizes mismatches created in the process of replication and uses some type of strand discrimination signal to selectively remove mismatched nucleotides present on the newly replicated strand of DNA (1-3). In most eukaryotic cells, there are two mismatch recognition complexes with different, but overlapping, specificities: (i) MutSα, a heterodimer of Msh2 and Msh6, recognizing base/base mismatches and small insertion/deletion loops; and (ii) MutSβ, a heterodimer of Msh2 and Msh3, recognizing both small and large loops (1-3). After recognition of a mispair by MutSα or MutSβ, completion of MMR requires association with proteins related to MutL, usually MutLα, which in yeast is a heterodimer of Mlh1 and Pms1 (1-3). DNA on the newly synthesized strand is then excised and the template strand rereplicated.The method of strand discrimination in eukaryotic MMR is still not solved, although major advances have recently been made. Several components of MMR are known to have association with proliferating cell nuclear antigen (PCNA), a sliding clamp that tracks with the replication fork (1-3). As would be predicted by that model, it has recently been shown that MMR is temporally coupled to replication (4) and that one pathway of MutSα-dependent MMR is through recruitment by a PCNA-Msh6 interaction (5). Whatever signals are used for strand discrimination are presumably lost as replication proceeds.To study various aspects of MMR, we have used single-stranded oligonucleotides (oligos) to introduce specific mispairs into Saccharomyces cerevisiae chromosomal DNA. We have shown previously that oligos can be introduced into cells by electroporation and can correct frame-shift mutations in LYS2 and that this process is inhibited by MMR (6). Our results are most consistent with a mechanism in which the oligo anneals to either the leading or lagging strand of replication at the replication fork, with subsequent extension. Mispairs created by the oligos are recognized by MMR, but those mispairs that escape MMR recognition create mutations in the next round of replication (6). For the experiments ...

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.

Previously published online as a Prion E-publication

DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair.