Sensitivity of a S. cerevisiae RAD27 deletion mutant to DNA-damaging agents and in vivo complementation by the human FEN-1 gene.

We have investigated the sensitivity to DNA-damaging agents of a strain of Saccharomyces cerevisiae containing a deletion of the RAD27 gene. The mutant strain is sensitive to a number of alkylating agents that modify DNA at a variety of positions, including one that produces primarily phosphotriesters. In contrast, the mutant strain is not sensitive to the oxidizing agent hydrogen peroxide. The introduction of a plasmid containing the FEN-1 gene (the human ortholog of the RAD27 gene) can substantially complement the sensitivity to alkylating agents observed in the mutant strain.

The chromatin structure of the GAL1 promoter forms independently of Reb1p in Saccharomyces cerevisiae.

Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces cerevisiae results from a network of interactions between transcription factors and chromatin. In this study we used footprinting procedures to characterize these interactions in vivo. DNase I analysis of the GAL1 upstream activating sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during growth in galactose, and also revealed binding of the Reb1 protein (Reb1p) during growth in glucose. In addition, we mapped to nucleotide resolution a positioned nucleosome that, in the inactive promoter, packages DNA between the UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements nucleosome free. The nucleosome footprint was lost when the promoter was activated. Surprisingly, mutation of the Reb1p binding site had no effect on nucleosome positioning or on the kinetics or extent of activation or repression of either the GAL1 or GAL10 promoters under any of the conditions assayed.

Recovery of RNA polymerase II synthesis following DNA damage in mutants of Saccharomyces cerevisiae defective in nucleotide excision repair.

We have measured the kinetics of the recovery of mRNA synthesis in the inducible GAL10 and RNR3 genes after exposure of yeast cells to ultraviolet (UV) radiation. Such recovery is abolished in mutant strains defective in nucleotide excision repair (NER) of DNA, including a rad23 mutant. Mutants defective in the RAD7 or RAD16 genes, which are required for the repair of the non-transcribed strand but not the transcribed strand of transcriptionally active genes, show slightly faster recovery of RNA synthesis than wild-type strains. A strain deleted of the RAD26 gene, which is known to be required for strand-specific NER in yeast, manifested delayed recovery of mRNA synthesis, whereas a rad28 mutant, which does not show defective strand-specific repair, showed normal kinetics of recovery. Measurement of the recovery of expression of selected individual yeast genes by Northern analysis following exposure of cells to UV radiation apparently correlates directly with the capacity of cells for strand-specific NER.

Interactions involving the human RNA polymerase II transcription/nucleotide excision repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group B (CSB) protein.

The human basal transcription factor TFIIH plays a central role in two distinct processes. TFIIH is an obligatory component of the RNA polymerase II (RNAP II) transcription initiation complex. Additionally, it is believed to be the core structure around which some if not all the components of the nucleotide excision repair (NER) machinery assemble to constitute a nucleotide excision repairosome. At least two of the subunits of TFIIH (XPB and XPD proteins) are implicated in the disease xeroderma pigmentosum (XP). We have exploited the availability of the cloned XPB, XPD, p62, p44, and p34 genes (all of which encode polypeptide subunits of TFIIH) to examine interactions between in vitro-translated polypeptides by co-immunoprecipitation. Additionally we have examined interactions between TFIIH components, the human NER protein XPG, and the CSB protein which is implicated in Cockayne syndrome (CS). Our analyses demonstrate that the XPB, XPD, p44, and p62 proteins interact with each other. XPG protein interacts with multiple subunits of TFIIH and with CSB protein.

The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH.

The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype. CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes. The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf. We report the cloning of the CSA cDNA, which can encode a WD repeat protein. Mutations in the cDNA have been identified in CS-A cell lines. CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH. These observations suggest that the products of the CSA and CSB genes are involved in transcription.

Characterization of a mutant strain of Saccharomyces cerevisiae 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 radiation-sensitive 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.

GAL4 disrupts a repressing nucleosome during activation of GAL1 transcription in vivo.

Photofootprinting in vivo of GAL1 reveals an activation-dependent pattern between the UASG and the TATA box, in a sequence not required for transcriptional activation by GAL4. The pattern results from a nucleosome whose position depends on sequences within the UASG. In the wild-type gene, activation by GAL4 and derivatives disrupts this nucleosome. This activity is independent of interactions with DNA-bound core transcription factors and is proportional to the strength of the activator. Presence of the nucleosome correlates with low basal transcription levels under various conditions, suggesting a role in limiting basal expression. We propose a role for the GAL4 activation domain in displacing a nucleosome and suggest that this is part of the mechanism by which GAL4 activates transcription in vivo.