The NER protein Rad33 shows functional homology to human Centrin2 and is involved in modification of Rad4.

In the yeast Saccharomyces cerevisiae the Rad4-Rad23 complex is implicated in the initial damage recognition of the Nucleotide Excision Repair (NER) pathway. NER removes a variety of lesions via two subpathways: Transcription Coupled Repair (TCR) and Global Genome Repair (GGR). We previously showed that the new NER protein Rad33 is involved in both NER subpathways TCR and GGR. In the present study we show UV induced modification of Rad4 that is strongly increased in cells deleted for RAD33. Modification of Rad4 in rad33 cells does not require the incision reaction but is dependent on the TCR factor Rad26. The predicted structure of Rad33 shows resemblance to the Centrin homologue Cdc31. In human cells, Centrin2 binds to XPC and is involved in NER. We demonstrate that Rad4 binds Rad33 directly and via the same conserved amino acids required for the interaction of XPC with Centrin2. Disruption of the Rad4-Rad33 interaction is sufficient to enhance the modification of Rad4 and results in a repair defect similar to that of a rad33 mutant. The current study suggests that the role of Rad33 in the Rad4-Rad23 complex might have parallels with the role of Centrin2 in the XPC-HHR23B complex.

Rad33, a new factor involved in nucleotide excision repair in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the Rad4-Rad23 complex is involved in initial damage recognition and responsible for recruiting the other NER proteins to the site of the lesion. The Rad4-Rad23 complex is essential for both NER subpathways, Transcription Coupled Repair (TCR) and Global Genome Repair (GGR). Previously, we reported on the role of the Rad4 homologue YDR314C in NER. YDR314C is essential for preferential repair of the transcribed strand in RNA pol I transcribed rDNA. In large scale interaction studies it was shown that YDR314C physically interacts with a small protein encoded by the ORF YML011C. In the present study we show that YML011C is involved in NER and we propose to designate the YML011C ORF RAD33. Cells deleted for RAD33 display intermediate UV sensitivity that is epistatic with NER. Strand specific repair analysis shows that GGR in RNA pol II transcribed regions is completely defective in rad33 mutants whereas TCR is still active, albeit much less efficient. In RNA pol I transcribed rDNA both GGR and TCR are fully dependent on Rad33. We show that in both rad23 and rad33 cells Rad4 and YDR314C protein levels are significantly reduced. The homology of YDR314C to Rad4, together with the similar relation of both proteins to Rad33 prompted us to propose RAD34 as name for the YDR314C gene. Although the rad23rad33 double mutant is considerably more UV sensitive than a rad23 or rad33 single mutant, deletion of RAD33 in a rad23 background does not lead to a further reduction of Rad4 or Rad34 protein levels. This suggests that the role of Rad33 is not solely the stabilization of Rad4 and Rad34 but that Rad33 has an additional role in NER.

The Rad4 homologue YDR314C is essential for strand-specific repair of RNA polymerase I-transcribed rDNA in Saccharomyces cerevisiae.

Summary The Saccharomyces cerevisiae protein Rad4 is involved in damage recognition in nucleotide excision repair (NER). In RNA polymerase II-transcribed regions Rad4 is essential for both NER subpathways global genome repair (GGR) and transcription coupled repair (TCR). In ribosomal DNA (rDNA), however, the RNA polymerase I-transcribed strand can be repaired in the absence of Rad4. In Saccharomyces cerevisiae the YDR314C protein shows homology to Rad4. The possible involvement of YDR314C in NER was studied by analysing strand-specific cyclobutane pyrimidine dimer (CPD) removal in both RNA pol I- and RNA pol II-transcribed genes. Here we show that the Rad4-independent repair of rDNA is dependent on YDR314C. Moreover, in Rad4 proficient cells preferential repair of the transcribed strand of RNA pol I-transcribed genes was lost after deletion of YDR314C, demonstrating that Rad4 cannot replace YDR314C. CPD removal from the RNA pol II-transcribed RPB2 gene was unaffected in ydr314c mutants. We conclude that the two homologous proteins Rad4 and YDR314C are both involved in NER and probably have a similar function, but operate at different loci in the genome and are unable to replace each other.

Anticancer drug resistance induced by disruption of the Saccharomyces cerevisiae NPR2 gene: a novel component involved in cisplatin- and doxorubicin-provoked cell kill.

The therapeutic potential of antitumor drugs is seriously limited by the manifestation of cellular drug resistance. We used the budding yeast Saccharomyces cerevisiae as a model system to identify novel mechanisms of resistance to one of the most active anticancer agents, cisplatin. We pinpointed NPR2 (nitrogen permease regulator 2) as a gene whose disruption conferred resistance to cisplatin. In addition, we observed a 4-fold cross-resistance of yeast npr2Delta cells (i.e., cells from which the NPR2 gene had been disrupted) to the anticancer drug doxorubicin, in combination with hypersensitivity to cadmium chloride. Furthermore, npr2Delta cells displayed unaltered cellular cisplatin and doxorubicin accumulation and showed an enhanced rate of spontaneous mutation compared with the isogenic parent. These data indicate that the npr2Delta phenotype overlaps that of the sky1Delta cells that we characterized previously (Mol Pharmacol 61:659-666, 2002). Therefore, we generated yeast npr2Delta sky1Delta double-knockout cells and performed clonogenic survival assays for cisplatin and doxorubicin, which revealed that NPR2 and SKY1 (SR-protein-specific kinase from budding yeast) are epistatic. The double-knockout strain was just as resistant to cisplatin and doxorubicin as the single-knockout strain that was most resistant to either drug. In conclusion, we identified NPR2 as a novel component involved in cell kill provoked by cisplatin and doxorubicin, and our data support the hypothesis that NPR2 and SKY1 may use mutual regulatory routes to mediate the cytotoxicity of these anticancer drugs.