Synergistically interacting gene mutations reveal buffering relationships that provide growth homeostasis through their compensation of one another. This analysis in Saccharomyces cerevisiae revealed genetic modules involved in tricarboxylic acid cycle regulation (RTG1, RTG2, RTG3), threonine biosynthesis (HOM3, HOM2, HOM6, THR1. THR4), amino acid permease trafficking (LST4, LST7), and threonine catabolism (GLY1). These modules contribute to a molecular circuit that regulates threonine metabolism and buffers deficiency in deoxyribonucleotide biosynthesis. Phenotypic, genetic, and biochemical evidence for this buffering circuit was obtained through analysis of deletion mutants, titratable alleles of ribonucleotide reductase genes, and measurements of intracellular deoxyribonucleotide pool concentrations. This circuit provides experimental evidence, in eukaryotes, for the presence of a high-flux backbone of metabolism, which was previously predicted from in silico modeling of global metabolism in bacteria. This part of the high-flux backbone appears to buffer deficiency in ribonucleotide reductase by enabling a compensatory increase in de novo purine biosynthesis that provides additional rate-limiting substrates for dNTP production and DNA synthesis. Hypotheses regarding unexpected connections between these metabolic pathways were facilitated by genome-wide but also highly quantitative phenotypic assessment of interactions. Validation of these hypotheses substantiates the added benefit of quantitative phenotyping for identifying subtleties in gene interaction networks that modulate cellular phenotypes. © 2007 by The National Academy of Sciences of the USA.