Abstract
In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ–DNA–PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ’s activity in replicating the human genome.
Highlights
In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation
The recently published cryo-EM structure of Saccharomyces cerevisiae (Sc) heterotrimeric Pol δ bound to (P/T) DNA elucidated the interactions between the catalytic subunit (Pol[3], homologous to human p125) and the two regulatory subunits (Pol[31] and Pol[32], homologous to human p50 and p66, respectively), showing a unique molecular arrangement[23]; the p12 subunit is absent in Sc Pol δ
We propose that dynamic interactions among PCNA loops and both the thumb and the C-terminal domain (CTD) of p125 may keep PCNA in a position that is competent for DNA polymerization
Summary
DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. The critical region of the CTD interacting with PCNA, encompassing the C-terminus of the thumb domain and the CysA motif, was invisible in the cryo-EM map of the isolated yeast holoenzyme[23], while it becomes visible in the human processive complex
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