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

Phenotypic characterization of the mutants.

<p>(A) Growth of the strains at different temperatures. Approximately 100 cells of each strain were plated on rich medium and the plates were incubated at the indicated temperatures for 2 days. (B) Sensitivity of the strains to HU. Ten fold serial dilutions of the indicated strains were spotted on YPD plates containing the given amount of HU. (C) Sensitivity of the strains to DNA damaging agents. Cells were spotted as described for (B) on plates either exposed to the indicated UV dose, or containing the indicated amount of MMS. For (B) and (C) several HU, UV, and MMS doses were applied, but only plates with the most appropriate doses are presented. (D) Quantitative assays of UV and MMS induced killing of the indicated strains. The results represent the average of three experiments. Standard deviations are indicated.</p

The positions of the mutated amino acids in the structure of PCNA.

<p>(A) Bar diagram of yeast PCNA. The positions of the altered amino acids, and of the IDCL are indicated. (B) The positions of the mutated amino acids at the subunit interface are shown in different colors in a schematic ribbon diagram representing the three dimensional structure of the PCNA trimer (source: Krishna, T.S.R at al. from RCSB PDB, PDB ID:1PLR) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161307#pone.0161307.ref007" target="_blank">7</a>]. The monomers are depicted in different colors.</p

Percentage of cells displaying the large budded (dumbbell) morphology of yeast strains expressing the PCNA variants.

<p>Percentage of cells displaying the large budded (dumbbell) morphology of yeast strains expressing the PCNA variants.</p

In the II99,100AA PCNA mutant strain the <i>REV3</i> branch of translesion synthesis is inactivated.

<p>(A-B) The II99,100AA PCNA mutant shows epistasis with <i>RAD18</i> upon UV treatment. (C-D) The II99,100AA PCNA mutant affects the Rev3 pathway. On (A) and (C) ten fold serial dilutions of cells with the indicated genotypes were spotted on plates and exposed to UV doses indicated on the right. (B) and (D) show quantitative analysis of UV-survival of the indicated strains. (E) UV-induced mutagenesis is abolished in the II99,100AA PCNA mutant strain. Forward mutation rates at the <i>CAN1</i> locus were determined after exposing the cells to the indicated UV doses. (B), (D), and (E) represent the average of three experiments. Standard deviations are indicated. P-values representing the significance of difference are also shown on (E). *:p<0.05 ** p<0.01, *** p<0.001 ns: no statistical difference.</p

Spontaneous mutation rates in yeast strains expressing the PCNA variants.

<p>Spontaneous mutation rates in yeast strains expressing the PCNA variants.</p

The II181,182AA PCNA behaves like wild type in its interaction with Rad54 <i>in vitro</i>.

<p>GST-Rad54 (3 μg) bound to glutathione-Sepharose beads was incubated with purified wild type (left panel), or II181,182AA mutant PCNA (right panel) (5–5 μg). After washing bound proteins were eluted with glutathione. Aliquots of each sample, taken from the input (I), from the unbound fraction (U), from the last wash (W), and from the glutathione-eluted proteins (E), were analyzed on 10% SDS polyacrylamide gel.</p

Complex formation between PCNA and Rev1.

<p>(A) GST-pull-down assay with Rev1-PAD and wild type, or II99,100AA PCNA. GST-Rev1-PAD (5 μg) immobilized on glutathione-Sepharose beads was incubated with purified wild type (left panel), or II99,100AA mutant PCNA (right panel) (5–5 μg). After washing bound proteins were eluted with glutathione. Aliquots of each sample, taken from the input (I), from the unbound fraction (U), from the last wash (W), and from the glutathione-eluted proteins (E), were analyzed on 10% SDS polyacrylamide gel. (B) GST-pull-down with Rev1 (5 μg) and wild type, or II99,100AA mutant PCNA (3–3 μg). Experiments were carried out as described for (A), but GST-Rev1 was used instead of GST-Rev1-PAD, and elution was achieved by PreScission protease cleavage of Rev1 from GST. The position of the cleaved Rev1 without the GST tag in the elution fraction is also marked.</p

The II181,182AA PCNA mutations affect HR differently based on the nature of the treatment of the cells.

<p>(A) The II181,182AA PCNA mutant is epistatic to both <i>RAD52</i> and <i>RAD54</i> upon UV-treatment. (B) Quantitative analysis of UV-survival of the <i>rad52 pol30-II181</i>,<i>182AA</i> double mutant. (C) X-ray (D) bleomycin (E) HU sensitivity of the <i>rad52 pol30-II181</i>,<i>182AA</i> double mutant strain. In panels (A), (C), (D), and (E) ten fold serial dilutions of strains with the indicated genotypes were spotted on YPD plates containing the given amount of bleomycin, or HU, or irradiated with the indicated UV or X-ray doses.</p