Coordination of Primer Initiation Within the Catalytic Domain of Human PrimPol

•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.