Abstract Our research is focused on understanding how the local chromatin environment regulates DNA-templated pro- cesses like transcription and DNA replication. We have developed and pioneered the use of factor-agnostic approaches to map chromatin occupancy at nucleotide resolution across the S. cerevisiae genome. We have used this approach to provide mechanistic insights into how the local chromatin environment mediates origin se- lection and activation. In order to identify the chromatin changes that occur with helicase activation at the onset of S-phase, we depleted cells of functional DNA polymerase alpha (Pol a) to prevent the priming of DNA synthesis. Activation of the helicase in the absence of priming not only resulted in the local disruption of chromatin, but also resulted in the uncoupling of the helicase from DNA synthesis and the unwinding of approximately 1 kb of DNA surrounding each activated origin. We will identify the mechanism(s) which regulate helicase progression in the absence of DNA replication with a focus on sequence, topological and rad53-mediated signaling. A conse- quence of the helicase traveling away from the origin in each direction and stalling is that upon restoration of Pol a priming, the helicase is oriented to travel away from the origin and thus will leave an unreplicated gap. These un- replicated gaps are a unique molecular intermediate that are analogous to intermediates predicted to occur from termination defects and provide a unique opportunity to identify factors involved in their resolution. We will also examine the role of specific histone chaperones in the spatiotemporal deposition of nucleosomes behind the DNA replication fork and how specific genomic features like active transcription may impact the maturation process in a locus specific manner. Finally, we have generated chromatin occupancy profiles for 201 yeast deletion mutants representing non-essential transcription factors and chromatin remodelers. Combining the chromatin occupancy profiles with gene expression data, we will be able to generate gene regulatory networks that are more accurately able to define direct and indirect targets.