The Saccharomyces cerevisiae RRM3 gene encodes a 5' to 3' DNA helicase. While replication of most of the yeast genome was not dependent upon Rrm3p, in its absence, replication forks paused and often broke at an estimated 1400 discrete sites, including tRNA genes, centromeres, inactive replication origins, and transcriptional silencers. These replication defects were associated with activation of the intra-S phase checkpoint. Activation of the checkpoint was critical for viability of rrm3Delta cells, especially at low temperatures. Each site whose replication was affected by Rrm3p is assembled into a nonnucleosomal protein-DNA complex. At tRNA genes and the silent mating type loci, disruption of these complexes eliminated dependence upon Rrm3p. These data indicate that the Rrm3p DNA helicase helps replication forks traverse protein-DNA complexes, naturally occurring impediments that are encountered in each S phase.