The eukaryotic cell cycle is a cascade of highly complex processes that must occur with striking temporal and spatial precision. This degree of complexity necessitates the existence of a sophisticated regulatory network capable not only of coordinating these events, but also of recognizing and correcting mistakes that occur during these complex processes. The infrastructured of these regulatory circuits will be probed in Saccharomyces cerevisiae using the genes encoding the enzyme ribonucleotide reductase, RNR1, 2, and 3, as tools. RNR1 and RNR2 are cell cycle- regulated, expressed maximally in S phase, and all three genes are transcriptionally induced in response to treatment with agents that damage DNA or block DNA replication. The cell cycle-regulation of RNR1 will be investigated by deletion and subcloning analysis to determine the sequences necessary and sufficient for cell cycle-regulation. Trans-acting factors that interact with these sequences will be examined with the long-term goal of isolating the genes involved in this regulation. In addition, induction of transcription of the RNR1 gene after passing the start of the cell cycle is dependent on protein synthesis. This dependency will be further investigated using known cell cycle mutants and differential cDNA screens to look for transcripts induced after Start, but prior to the protein synthesis block. Inhabitation of ribonucleotide reductase activity by the specific inhibitor hydroxyurea (HU) causes cell cycle arrest in S-phase that is independent of the RAD9 gene. Genes involved in this response to HU will be isolated by screening for HU-sensitive mutations that have an """"""""anti-CDC"""""""" phenotype with respect to HU-induced cell cycle arrest. Genes of interest will be isolated by complementation of these mutations. Analysis of this pathway will shed light on a critical regulatory circuit of the eukaryotic cell cycle that coordinate DNA replication with progression into G2 and M. The pathway that senses DNA damage and induces RNR expression will be investigated by the isolation of cis- and trans-mutations that alter the ability of the cell to properly regulate the RNR2 and RNR3 genes in response to DNA damage. Genes involved in this regulation will be isolated by complementation. The ability of an organism to sense and respond to damage to its genetic material is central to its ability to adapt to environmental stress and to survive. This regulatory circuit is likely to be conserved among eukaryotes and may shed light on the ability of higher eukaryotes to sense and respond to DNA damage. A dual purpose gamma-based plasmid expression vector will be constructed that is capable of regulated expression of genes in E. coli and yeast. Libraries constructed in this vector will be used to isolate yeast genes encoding sequence-specific DNA-binding proteins of interest by genetic selection in E. coli. These libraries will also be used in yeast to identify new cell division cycle (CDC) genes by screening for clones which when expressed, result in dominant lethality and arrest with a CDC phenotype.
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