The mRNA and protein expression in Saccharomyces cerevisiae cultured in rich or minimal media was analyzed by oligonucleotide arrays and quantitative multidimensional protein identification technology. The overall correlation between mRNA and protein expression was weakly positive with a Spearman rank correlation coefficient of 0.45 for 678 loci. To place the data sets in a proper biological context, a clustering approach based on protein pathways and protein complexes was implemented. Protein expression levels were transcriptionally controlled for not only single loci but for entire protein pathways (e.g., Met, Arg, and Leu biosynthetic pathways). In contrast, the protein expression of loci in several protein complexes (e.g., SPT, COPI, and ribosome) was posttranscriptionally controlled. The coupling of the methods described provided insight into the biology of S. cerevisiae and a clustering strategy by which future studies should be based.
In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G 1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G 1 cells. This G 1 -phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G 1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.
A competitive growth assay has been used to identify yeast genes involved in the repair of UV-or MMS-induced DNA damage. A collection of 2,827 yeast strains was analyzed in which each strain has a single ORF replaced with a cassette containing two unique sequence tags, allowing for its detection by hybridization to a high-density oligonucleotide array. The hybridization data identify a high percentage of the deletion strains present in the collection that were previously characterized as being sensitive to the DNAdamaging agents. The assay, and subsequent analysis, has been used to identify six genes not formerly known to be involved in the damage response, whose deletion renders the yeast sensitive to UV or MMS treatment. The recently identified genes include three uncharacterized ORFs, as well as genes that encode protein products implicated in ubiquitination, gene silencing, and transport across the mitochondrial membrane. Epistatsis analysis of four of the genes was performed to determine the DNA damage repair pathways in which the protein products function. DNA is a labile molecule that can undergo spontaneous hydrolysis or modification by physical and chemical agents within the cellular environment (1). Modified DNA must be rapidly recognized and efficiently repaired, thus both prokaryotes and eukaryotes have evolved complex surveillance and repair mechanisms. The response to DNA damage has been well characterized in Saccharomyces cerevisiae through the isolation of mutants that are hypersensitive to specific DNA damaging agents (1). These mutation studies led to the definition of three groups of proteins involved in different types of DNA repair, termed the RAD3, RAD52, and RAD6 epistasis groups, based on phenotypic sensitivity to UV, ionizing radiation, or both.The cellular DNA damage response depends on the type of damage incurred. The UV response is largely mediated by two epistasis groups, the RAD3 group, which includes genes of the nucleotide excision and repair pathway, and the RAD6 group, encoding proteins involved in postreplication repair and damage-induced mutagenesis. Treating cells with the methylating agent methyl methanesulfonate (MMS) results in alkylated DNA, which is poorly replicated by DNA polymerases in vitro and in vivo (1, 2), and must be efficiently repaired. Base excision repair proteins (3), as well as proteins encoded by the MEC1 and RAD53 genes (2), and the RAD6 and the RAD52 epistasis group genes (4), have all been implicated in the response to MMS damage. Although excision repair and recombination repair pathways are relatively well understood, much less is known about the RAD6 mediated pathway. The response to both UV irradiation and MMS methylation likely involve additional genes, especially of the RAD6 pathway, which remain to be identified.Toward this end, we have used a collection of S. cerevisiae deletion strains, wherein each strain has had an individual gene replaced by a unique DNA sequence, to phenotypically screen the yeast genome for proteins involved in the DNA da...
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