Abstract
DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication and genome maintenance, essential for lagging strand synthesis, leading strand initiation, and DNA repair. While human Pol δ exhibits high activity and processivity in its holoenzyme form complexed with proliferating cell nuclear antigen (PCNA), it shows minimal DNA synthesis activity without PCNA, the molecular basis of which remains unclear. Here, we present the cryo-EM structure of the apo-form human Pol δ, comprising the catalytic subunit p125 and regulatory subunits p66, p50, and p12, at an overall resolution of 3.65 Å. We identified an acidic α-helix at the N terminus of p125, which occupies the single-stranded DNA-binding cavity within the polymerase domain in the apo-form Pol δ. This interaction likely inhibits DNA binding in the absence of PCNA, explaining the low activity of apo-form Pol δ. The acidic α-helix is absent in yeast Pol δ, providing a molecular explanation for species-specific differences in PCNA-independent Pol δ activity. These findings provide critical insights into the regulatory mechanisms of Pol δ and its reliance on PCNA for efficient DNA synthesis.