Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

The authors would like to thank Mark Hochstrasser for the MHY500 strain and Yasushi Saeki for the proteasome mutant and the corresponding wild-type strains. We also thank Sz. Minorits for technical assistance. This publication was also supported by the Dr. Rollin D. Hotchkiss Foundation. Funding: Wellcome Trust, 070247/Z/03/A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome-mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

Acknowledgments We thank Szilvia Minorits for technical assistance. I.U. conceived and designed the project and wrote the manuscript. All authors participated in designing and performing the experiments, and analyzing the results. The authors declare no competing financial interests. This work was also supported by a grant from the National Research, Development and Innovation Office GINOP-2.3.2-15-2016-00001. Funding: This work was supported by Hungarian Science Foundation Grant OTKA 109521 and National Research Development and Innovation Office GINOP-2.3.2-15-2016-00001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

TAP of Rad5 and its complexes.

<p>The final elution fractions of TAP purification of whole cell extracts from control and MMS-treated cultures were analyzed by SDS PAGE. Purified proteins are designated on the right, and the molecular mass markers are indicated on the left. The asterisk denotes a nonspecific band.</p

UV-dose–dependent degradation of Pol3.

<p>Cultures of <i>mms2</i> cells were synchronized by α-factor and irradiated with increasing doses of UV, as indicated. After released back to growth, 1 ml of cells was collected at the indicated time points, and cell extracts were analyzed by Western blotting. Anti-HA detected HA-tagged Pol3, and PGK served as a loading control. The level of Pol3 relative to PGK is shown at the bottom of each panel.</p

<i>DEF1</i> participates in the <i>REV3</i> branch of the <i>RAD6</i>-governed DNA damage tolerance.

<p>(A–E) Epistatic analysis of <i>DEF1</i> with mutants of the different branches of the <i>RAD6</i> pathway upon UV irradiation. Standard deviations are indicated. (F–J) Epistatic analysis of the same mutants upon MMS treatment. (K, L) Genetic interactions of <i>RAD30</i> with <i>MMS2</i> and <i>REV3</i> upon MMS treatment. All experiments were repeated at least three times.</p

Pol3 degradation depends on <i>RAD6</i> and <i>DEF1</i>.

<p>Cultures were synchronized by α-factor, UV-irradiated with 150 J/m², and released back to growth media. Proteins from whole cell extracts, prepared from 1 ml of cell culture collected at the indicated time points after UV treatment, were analyzed by Western blotting. Anti-HA antibody was used to detect HA-tagged Pol3 (A to E), Pol31 (F), or Pol32 (G). Cell cycle progression was monitored by Clb2 cyclin levels, and PGK served as a loading control. The level of Pol3 relative to PGK is shown at the bottom of each panel.</p

DNA-damage–induced mutagenesis is abolished in <i>def1</i> deletion mutants.

<p>Forward mutation rates at the <i>CAN1</i> locus were determined after UV treatment. Where indicated, <i>def1</i> deletion was complemented by wild-type <i>DEF1</i> expressed under the control of the ADH1 promoter on a centromeric plasmid. The standard deviation is indicated above each bar. Experiments were repeated three times.</p