Chromosomal DNA elements are organized into spatial domains within the eukaryotic nucleus. For example, sites actively undergoing DNA replication, high-level transcription, and repair of double-strand breaks coalesce into foci, although the significance and mechanisms giving rise to these dynamic structures are poorly understood. In S. cerevisiae, replication origins exhibit characteristic subnuclear localizations prior to S phase that anticipate their initiation timing and/or efficiency during S phase: origins found within the nuclear interior in G1 phase initiate early and efficiently in S phase while origins found associated with the nuclear periphery in G1 phase initiate later and less efficiently. In this study, we link interior localization of replication origins in G1 phase with Fkh1 activity, which is required for their early replication timing. Using a Fkh1-dependent origin relocalization assay, we determine that execution of Dbf4-dependent kinase function, including Cdc45 loading, results in dynamic relocalization of a replication origin from the nuclear periphery to the interior in G1 phase. Origin mobility increases substantially with Fkh1-driven relocalization. These findings provide novel molecular insight into the mechanisms that govern dynamics and spatial organization of DNA replication origins and possibly other functional DNA elements.