The Compact Chromatin Structure of a Ty Repeated Sequence Suppresses Recombination Hotspot Activity in Saccharomyces cerevisiae

Recombination between repeated DNA sequences can have drastic consequences on the integrity of the genome. Repeated sequences are abundant in most eukaryotes, yet the mechanism that prevents recombination between them is currently unknown. Ty elements, the main family of dispersed repeats in Saccharomyces cerevisiae, exhibit low levels of exchange. Other regions in the genome have relatively high rates of meiotic recombination (hotspots). We show that a Ty element adjacent to the HIS4 recombination hotspot substantially reduces its activity, eliminating local DSB formation. We demonstrate that the Ty has a closed (nuclease-insensitive) chromatin configuration that is also imposed on the flanking DNA sequences. The compact chromatin structure is determined by sequences at the N terminus of the Ty. Increased binding of the Rap1 protein to the hotspot restores both open chromatin conformation and DSB formation. The chromatin configuration of Ty elements precludes initiation of recombination, thus preventing potentially lethal exchanges between repeated sequences

Structural Insight into Regulation of the Proteasome Ub-Receptor Rpn10

Ubiquitylation is a posttranslational modification that determines protein fate. The ubiquitin code is written by enzymatic cascades of E1 and E2 and E3 enzymes. Ubiquitylation can be edited or erased by deubiquitylating enzymes. Ub-receptors are proteins that read and decipher the ubiquitin codes into cellular response. They harbor a ubiquitin-binding domain and a response element. Interestingly, Ub-receptors are also regulated by ubiquitylation and deubiquitylation. However, until recently, the molecular details and the significance of this regulation remained enigmatic. Rpn10 is a Ub-receptor that shuttles ubiquitylated targets to the proteasome for degradation. Here we review recent data on Rpn10, with emphasis on its regulation by ubiquitylation

Proteasome Nuclear Activity Affects Chromosome Stability by Controlling the Turnover of Mms22, a Protein Important for DNA Repair

To expand the known spectrum of genes that maintain genome stability, we screened a recently released collection of temperature sensitive (Ts) yeast mutants for a chromosome instability (CIN) phenotype. Proteasome subunit genes represented a major functional group, and subsequent analysis demonstrated an evolutionarily conserved role in CIN. Analysis of individual proteasome core and lid subunit mutations showed that the CIN phenotype at semi-permissive temperature is associated with failure of subunit localization to the nucleus. The resultant proteasome dysfunction affects chromosome stability by impairing the kinetics of double strand break (DSB) repair. We show that the DNA repair protein Mms22 is required for DSB repair, and recruited to chromatin in a ubiquitin-dependent manner as a result of DNA damage. Moreover, subsequent proteasome-mediated degradation of Mms22 is necessary and sufficient for cell cycle progression through the G2/M arrest induced by DNA damage. Our results demonstrate for the first time that a double strand break repair protein is a proteasome target, and thus link nuclear proteasomal activity and DSB repair

The Complete Spectrum of Yeast Chromosome Instability Genes Identifies Candidate CIN Cancer Genes and Functional Roles for ASTRA Complex Components

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease

The Elg1 Clamp Loader Plays a Role in Sister Chromatid Cohesion

Mutations in the ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring

Subtle Alterations in PCNA-Partner Interactions Severely Impair DNA Replication and Repair

Dynamic switching of PCNA-partner interactions is essential for normal DNA replication and repair in yeast

Inactive Proteasomes Routed to Autophagic Turnover Are Confined within the Soluble Fraction of the Cell

Previous studies demonstrated that dysfunctional yeast proteasomes accumulate in the insoluble protein deposit (IPOD), described as the final deposition site for amyloidogenic insoluble proteins and that this compartment also mediates proteasome ubiquitination, a prerequisite for their targeted autophagy (proteaphagy). Here, we examined the solubility state of proteasomes subjected to autophagy as a result of their inactivation, or under nutrient starvation. In both cases, only soluble proteasomes could serve as a substrate to autophagy, suggesting a modified model whereby substrates for proteaphagy are dysfunctional proteasomes in their near-native soluble state, and not as previously believed, those sequestered at the IPOD. Furthermore, the insoluble fraction accumulating in the IPOD represents an alternative pathway, enabling the removal of inactive proteasomes that escaped proteaphagy when the system became saturated. Altogether, we suggest that the relocalization of proteasomes to soluble aggregates represents a general stage of proteasome recycling through autophagy

Chapter Structural Insight into Regulation of the Proteasome Ub-Receptor Rpn10

Ubiquitylation is a posttranslational modification that determines protein fate. The ubiquitin code is written by enzymatic cascades of E1 and E2 and E3 enzymes. Ubiquitylation can be edited or erased by deubiquitylating enzymes. Ub-receptors are proteins that read and decipher the ubiquitin codes into cellular response. They harbor a ubiquitin-binding domain and a response element. Interestingly, Ub-receptors are also regulated by ubiquitylation and deubiquitylation. However, until recently, the molecular details and the significance of this regulation remained enigmatic. Rpn10 is a Ub-receptor that shuttles ubiquitylated targets to the proteasome for degradation. Here we review recent data on Rpn10, with emphasis on its regulation by ubiquitylation

Chapter Structural Insight into Regulation of the Proteasome Ub-Receptor Rpn10

Ubiquitylation is a posttranslational modification that determines protein fate. The ubiquitin code is written by enzymatic cascades of E1 and E2 and E3 enzymes. Ubiquitylation can be edited or erased by deubiquitylating enzymes. Ub-receptors are proteins that read and decipher the ubiquitin codes into cellular response. They harbor a ubiquitin-binding domain and a response element. Interestingly, Ub-receptors are also regulated by ubiquitylation and deubiquitylation. However, until recently, the molecular details and the significance of this regulation remained enigmatic. Rpn10 is a Ub-receptor that shuttles ubiquitylated targets to the proteasome for degradation. Here we review recent data on Rpn10, with emphasis on its regulation by ubiquitylation