Thioredoxin is a small protein which reduces protein disulfides. Possible roles for thioredoxin include but are not limited to: 1) action as an autocrine growth factor, 2) a regulator of hormone binding by steroid receptors, 3) regeneration of proteins inactivated by oxidation, 4) a role in interferon mediated growth arrest, and 5) part of the early pregnancy factor activity of pregnancy serum. As a principal hydrogen donor to ribonucleotide reductase, thioredoxin also has an important role in cellular and viral DNA replication. The broad objective of this application is to acquire new insights into the function of thioredoxin in cell growth and division. towards this goal the proposal combines biochemical and genetic approaches to understand the cellular response to thioredoxin deficiency in mutants of Saccharomyces cerevisiae. Deletion of two thioredoxin genes in yeast inhibits DNA synthesis, but cells are still viable. The cause of the slow rate of DNA synthesis is unknown. Since deoxyribonucleotide levels remain high, ribonucleotide reductase activity appears adequate. Besides a role in DNA replication other roles for thioredoxin may be masked by pathways functionally redundant with thioredoxin. The specific aims of this application are two-fold. First, to continue the study of DNA synthesis in the thioredoxin mutant. Second to identify and eliminate the thioredoxin alternative pathways, possibly uncovering additional functions for thioredoxin. As part of the first aim, the rate of DNA elongation will be measured in the thioredoxin mutant, and thioredoxin will be localized. The experiments will help evaluate two alternative hypothesis for the sub-optimal rates of DNA synthesis in the thioredoxin mutant. As part of the second aim, a thd1 mutant and the THD1 gene will be characterized. Thd1 mutants require thioredoxin for viability, suggesting that the alternative pathways are blocked. Based upon DNA sequence, THD1 encodes glutathione reductase. The sequence of the THD1 gene will be completed. Glutathione reductase, glutathione and other thiols will be assayed in the thdl mutant. If DNA synthesis is inhibited, deoxyribonucleotide pools will be measured. Also, the sensitivity of thd1 mutants to oxidants will be evaluated. Thioredoxin is essential in the thd1 mutants, so for the first time the isolation of a temperature-sensitive permissive temperature will reveal whether thioredoxin has other essential roles in yeast beyond a function in DNA synthesis. As part of the characterization of the trx1ts, nucleotide levels, thiol composition, protein and RNA synthesis, cell cycle parameters, and cell morphology will be examined. Finally thd75 mutants differ from thd1 mutants in that they require multiple copies of thioredoxin gene for survival. The gene complementing thd75 will be cloned and sequenced to determine if this uncommon mutant reveals novel functions for thioredoxin.