Recent advances in understanding bacterial and archaeoeukaryotic primases

•Primases are part of the replisome, a flexible and highly dynamic multiprotein complex at the replication fork.•Initiation of primer synthesis requires binding of three substrates that yield the dinucleotide/template hybrid further elongated by the primase.•Substrate binding involves synergistic interactions on the non-catalytic domain as exemplified by an archaeal homolog.•Beside replicative functions, primases such as PrimPol are a key factor for genome stability. DNA replication in all forms of life relies upon the initiation of synthesis on a single strand template by formation of a short oligonucleotide primer, which is subsequently elongated by DNA polymerases. Two structurally distinct classes of enzymes have evolved to perform this function, namely the bacterial DnaG-type primases and the Archaeal and Eukaryotic primases (AEP). Structural and mechanistic insights have provided a clear understanding of the role of the different domains of these enzymes in the context of the replisome and recent work sheds light upon primase-substrate interactions. We herein review the emerging picture of the primase mechanism on the basis of the structural knowledge obtained to date and propose future directions of this essential aspect of DNA replication.