Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

Human DNA polymerase delta (Pol delta) forms a holoenzyme complex with the DNA sliding clamp proliferating cell nuclear antigen (PCNA) to perform its essential roles in genome replication. Here, we utilize live-cell single-molecule tracking to monitor Pol delta holoenzyme interaction with the genome in real time. We find holoenzyme assembly and disassembly in vivo are highly dynamic and ordered. PCNA generally loads onto the genome before Pol delta. Once assembled, the holoenzyme has a relatively short lifetime on the genome, implying multiple Pol delta binding events may be needed to synthesize an Okazaki fragment. During disassembly, Pol delta dissociation generally precedes PCNA unloading. We also find that Pol delta p125, the catalytic subunit of the holoenzyme, is maintained at a constant cellular level, indicating an active mechanism for control of Pol delta levels in vivo. Collectively, our studies reveal that Pol delta holoenzyme assembly and disassembly follow a predominant pathway in vivo; however, alternate pathways are observed.