Yeast replication origins contain a DNA sequence element whose biological activity correlates with hypersensitivity to single-strand-specific nucleases in negatively supercoiled plasmids. By using two-dimensional gel electrophoresis of plasmid topoisomers, we demonstrate that thermodynamically stable origin unwinding accounts for the nuclease hypersensitivity and, furthermore, that increased thermal energy facilitates stable origin unwinding in vitro. In living cells, increased thermal energy can suppress origin mutations that raise the free-energy cost for unwinding the nuclease-hypersensitive element. Specifically, mutational defects in autonomously replicating sequence (ARS)-mediated plasmid replication are less severe in cells grown at 30 degrees C as compared to 23 degrees C. Our findings indicate that the energetics of DNA unwinding at the nuclease-hypersensitive element are biologically important. We call the nuclease-hypersensitive sequence the DNA unwinding element (DUE) and propose that it serves as the entry site for yeast replication enzymes into the DNA helix.