Structural and mechanistic insights into primer synthesis initiation by DNA primase

DNA primases synthesize short primers required for genome replication, yet the mechanism of initial dinucleotide formation remains poorly understood. Here, we investigate the primase encoded by the pRN1 plasmid from the thermoacidophile archaeon Sulfolobus islandicus, a minimal model for primer synthesis. Using nucleotide analogues to slow the reaction, we capture transient intermediates of dinucleotide formation. Structural NMR and modeling reveal that the ancillary domain simultaneously binds the DNA template and two initiating nucleotides. Unexpectedly, only the second nucleotide base-pairs with the template, whereas the first remains unpaired, inducing template-base flipping and linker interaction. This interaction promotes a closed conformation in which the second nucleotide moves from the initiation to the elongation site and the first forms a base pair in the initiation site, positioning both nucleotides for catalysis. These findings reveal a mechanism for template recognition, nucleotide assembly, and proofreading during primer initiation that is likely conserved among primases.