9728782 Merrill All eucaryotic cells possess a mechanism for inducing the transcription of a specific class of genes at the G1/S boundary of the cell cycle. In yeast, short cis-acting elements termed MCBs have been implicated in G1/S gene induction. Similar elements, termed E2F sites, have been implicated in higher eucaryotes. The genes are induced shortly after cells commit to the cell cycle, and the mechanism linking gene induction to the commitment decision has been the subject of increasingly intense research. In a genetic screen for mutations that allow MCB reporter gene expression in yeast lacking the MCB transcription factor Swi6, all thirty of the mutations isolated mapped to a single gene - TRRI - encoding thioredoxin reductase. Deleting TRRI or deleting the yeast thioredoxin genes TRXI and TRX2 also strongly activated MCB reporter gene expression. The trrl mutations did not increase the asynchronous cell levels or eliminate the periodicity of several endogenous MCB gene mRNAs, however, peak mRNA levels occurred earlier in the mutants. Conditions expected to oxidize thioredoxin (exposure to H202) induced MCB gene expression, whereas conditions expected to conserve reduced thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results indicate that thioredoxin oxidation contributes to MCB element activation. Thioredoxin oxidation may either inactivate a negative regulator of MCB activity, or activate a positive regulator. One goal of the project is to test the hypothesis that a transient episode of RNR-mediated oxidation of thioredoxin contributes to MCB gene activation at G1/S. RNR-mediated oxidation of thioredoxin neatly explains how gene induction could be coupled to replication initiation. The prediction that MCB gene induction should be blccked if DNA replication is inhibited will be tested using (-factor-synchronized dbf4 cells that cannot initiate replication at the nonpermissive temperature. The prediction that thioredoxin should become more oxidize d at G1/S will be tested in (-factor-synchronized dbf4 cells incubated at the permissive and nonpermissive temperature. The in vivo redox state of thioredoxin will be determined by a method involving immunoblotting after differential alkylation. As asynchronous cell measurements indicate the dNTP pool in yeast is only a small fraction of the size needed for genome replication, increased reduction of ribonucleotides must occur near the G1/S border. To determine whether increased ribonucleotide reduction is due to allosteric or non-allosteric mechanisms, RNR protein, RNA activity, and dNTP pools sizes will be measured in synchronized cells. In addition to testing a specific model for RNR involvement in transcriptional control at G1/S, the experiments will yield fundamental information about biochemical changes that occur during the cell cycle. A second goal of the research is to identify downstream mediators of the thioredoxin/MCB regulation pathway. Three specific proteins that are potential mediators of the thioredoxin effect on MCB activity will be evaluated by determining whether trr1 mutations activate MCB genes in yeast lacking the SW14, SKN7, or YAP1 genes. Once genetic approaches have identified a protein suspected of mediating the thioredoxin effect, in vitro biochemical experiments will investigate whether the protein has oxidation-prone thiols that are efficiently reduced by thioredoxin, and site-directed mutagenesis experiments will evaluate the effect of modifying specific cysteines on protein function in vivo. Several genes are preferentially expressed at about the time that cells begin replicating their DNA. The planned experiments test the idea that DNA replication itself is the switch that turns on gene expression. To replace the DNA precursors consumed by the replication process, the cell must rapidly reduce ribonucleotides to the deoxyribonucleotide form. The probable source of electrons during ribonucleotide reduction is thioredoxin - a protein that maintains other proteins in their reduced state. Thus, DNA replication may trigger an episode of thioredoxin oxidation, which in turn may oxidize control proteins that regulate transcription.
