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In humans, DNA polymerase δ (Pol δ) holoenzymes, comprised of Pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand DNA replication, initiation of leading strand DNA replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a coordinated process involving the major single strand DNA-binding protein complex, replication protein A (RPA), the processivity sliding clamp loader, replication factor C (RFC), PCNA, and Pol δ. Each of these factors interact uniquely with a P/T junction and most directly engage one another. Currently, the interplay between these macromolecular interactions is largely unknown. In the present study, novel Förster Resonance Energy Transfer (FRET) assays reveal that dynamic interactions of RPA with a P/T junction during assembly of a Pol δ holoenzyme and initiation of DNA synthesis maintain RPA at a P/T junction and accommodate RFC, PCNA, and Pol δ, maximizing the efficiency of each process. Collectively, these studies significantly advance our understanding of human DNA replication and DNA repair., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.1