Journal of Molecular Biology

To initiate primer synthesis, PrimPol coordinates nucleotides, DNA and metals.

Sequential substrate binding in the active site stabilises the initiation complex.

Dinucleotide formation is the rate-limiting step of primer synthesis.

PrimPol preferentially initiates synthesis upon 3′-RCC-5′ sequences.

The mechanism of de novo primer synthesis is conserved from bacteria to humans.

To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol’s active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol’s catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the ‘choreography’ of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.

Primase

PrimPol

CAPP

Prim-Pol

Priming

© 2023 The Author(s). Published by Elsevier Ltd.