Characterization of a mutant strain of \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene

We have constructed a strain of Saccharomyces cerevisiae with a deletion of the YKL510 open reading frame, which was initially identified in chromosome XI as a homolog of the RAD2 nucleotide excision repair gene (A. Jacquier, P. Legrain, and B. Dujon, Yeast 8:121–132, 1992). The mutant strain exhibits increased sensitivity to UV light and to the alkylating agent methylmethane sulfonate but not to ionizing radiation. We have renamed the YKL510 open reading frame the RAD27 gene, in keeping with the accepted nomenclature for radiationsensitive yeast mutants. Epistasis analysis indicates that the gene is in the RAD6 group of genes, which are involved in DNA damage tolerance. The mutant strain also exhibits increased plasmid loss, increased spontaneous mutagenesis, and a temperature-sensitive lethality whose phenotype suggests a defect in DNA replication. Levels of the RAD27 gene transcript are cell cycle regulated in a manner similar to those for several other genes whose products are known to be involved in DNA replication. We discuss the possible role of Rad27 protein in DNA repair and replication

NSC109268 potentiates cisplatin-induced cell death in a p53-independent manner

This article's study endeavors to determine if p53 is responsible for cisplatin sensitization by NSC109268. Results have implications for sensitivity of ovarian cancer cells to cisplatin independent of p53

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

2-Deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polζ (rev3 mutant), but were only marginally reduced in the absence of Polη (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polζ are required for the efficient bypass of abasic sites in yeast

Rad5 Template Switch Pathway of DNA Damage Tolerance Determines Synergism between Cisplatin and NSC109268 in <i>Saccharomyces cerevisiae</i>

<div><p>The success of cisplatin (CP) based therapy is often hindered by acquisition of CP resistance. We isolated NSC109268 as a compound altering cellular sensitivity to DNA damaging agents. Previous investigation revealed an enhancement of CP sensitivity by NSC109268 in wild-type <i>Saccharomyces cerevisiae</i> and CP-sensitive and -resistant cancer cell lines that correlated with a slower S phase traversal. Here, we extended these studies to determine the target pathway(s) of NSC109268 in mediating CP sensitization, using yeast as a model. We reasoned that mutants defective in the relevant target of NSC109268 should be hypersensitive to CP and the sensitization effect by NSC109268 should be absent or strongly reduced. A survey of various yeast deletion mutants converged on the Rad5 pathway of DNA damage tolerance by template switching as the likely target pathway of NSC109268 in mediating cellular sensitization to CP. Additionally, cell cycle delays following CP treatment were not synergistically influenced by NSC109268 in the CP hypersensitive <i>rad5Δ</i> mutant. The involvement of the known inhibitory activities of NSC109268 on 20S proteasome and phosphatases 2Cα and 2A was tested. In the CP hypersensitive <i>ptc2Δptc3Δpph3Δ</i> yeast strain, deficient for 2C and 2A-type phosphatases, cellular sensitization to CP by NSC109268 was greatly reduced. It is therefore suggested that NSC109268 affects CP sensitivity by inhibiting the activity of unknown protein(s) whose dephosphorylation is required for the template switch pathway. </p> </div

Influence of phosphatase deficiencies on NSC109268 in mediating cisplatin sensitization.

<p>Survival analyses of single, double and triple phosphatase gene deletion strains of yeast treated with CP, alone or in combination with NSC109268 (3 μM). Dose-response curves of YJK17 (WT), YJK26 (pph3∆), YJK24 (ptc2∆ptc3∆) and YJK70 (ptc2Δptc3Δpph3Δ) are shown. Surviving fractions are plotted as a function of CP dose, with or without NSC109268 (3 μM) present during treatment. Use of symbols is indicated in the figure.</p

Effect of sequential treatment with cisplatin and NSC109268 on yeast cell killing.

<p>Survival of logarithmic-phase haploid wild-type yeast (BY4741) was analyzed after pretreating cells with CP for 1 h, followed by NSC109268 (3 μM) for 1 h in CP-free PBS. Fractions of colony forming cells were plotted as a function of CP dose, with and without NSC109268 administration. </p

Effect of NSC109268 on cisplatin sensitivity in various deletion mutants.

<p>Strains of two different genetic backgrounds (BY4741, YJK17) were ranked by dose enhancement factors at 50% survival. Data were corrected for inactivation by NSC109268 alone. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077666#pone.0077666.s005" target="_blank">Table S1</a> for the putative role of gene products.</p

Effect of cisplatin and NSC109268 on survival and induced gene conversion/mitotic recombination in a diploid reporter strain.

<p>(A) Surviving fractions of colony forming cells, (B) frequency of TRP⁺ convertants, (C) frequency of red or pink sectored or pure clones of strain D7. Data were plotted as a function of CP dose, administered with or without added NSC109268 (8 µM). Values in B and C were corrected for spontaneous TRP⁺ and red/pink colony background frequencies.</p