ABSTRACT The accurate and efficient duplication of the eukaryotic genome requires the activation of multiple DNA replication origins distributed over the length of each chromosome. While each origin has the same basic purpose—to direct unwinding of the chromosomal DNA for the initiation of DNA synthesis—each must function within a distinct chromatin context. Despite the well known heterogeneity of chromatin, the field has little understanding of how the core origin binding proteins function within different chromatin contexts or whether origin DNA sequence requirements are influenced by chromatin. The work in the lab of Catherine Fox indicates that these issues are important for explaining why individual origins exhibit differences in how often (origin efficiency) and when (origin activation time) they function during S-phase. This origin functional diversity establishes a temporal pattern to chromosomal replication that is crucial to both cell differentiation and genome stability. The proposed research will define mechanisms that underlie origin functional diversity in the model organism Saccharomyces cerevisiae because the origin-binding proteins and many aspects of chromatin are conserved. In contrast to early models for how origin-specific functional differences in yeast are achieved, the Fox lab proposes that chromatin-mediated regulation of ORC-DNA interactions help establish origin functional diversity. The lab's research provides evidence that ORC-DNA binding in vivo is a target of both positive and negative regulation by chromatin, depending on context; the proposed research will examine the molecular mechanism of two evolutionarily conserved positive regulators. The experiments in this proposal will address a fundamental hypothesis that functionally relevant ORC-origin binding results from a combination of ORC-DNA and ORC-chromatin interactions that result in origin-specific ORC-DNA dynamics that contribute to the regulation of origin function in S-phase.