Phylogenetic and functional characterization of Asgard primases

Abstract Eukarya resemble Archaea in DNA replication. Analysis of the DNA replication machinery of Asgard archaea may provide a valuable test of the hypothesis of this phylum being the origin of Eukarya. Among the replication proteins, primase, which comprises the catalytic subunit PriS and the noncatalytic subunit PriL, synthesizes primers for extension by DNA polymerase. Here we show that Asgard primases fall into two major groups, denoted the Heimdall group and the Loki group, which are phylogenetically and structurally more closely related to eukaryotic primases and primases from non-Asgard archaea, respectively. Notably, like human PriL, PriL of the Heimdall group possesses an extra C-terminal domain, which, absent in archaeal PriL of the non-Heimdall group, presumably serves to enhance the stability of the conserved iron-sulfur cluster in PriL. We overproduced in Escherichia coli and purified the PriS and PriL subunits of the Heimdall group from the Candidatus Gerdarchaeota archaeon B18_G1. Biochemical characterization reveals that the B18_G1 primase is capable of primer synthesis and extension, using preferentially dNTPs as the substrates, as shown for primases from non-Asgard archaea, but, unlike the non-Asgard archaeal primases, it produces short primers, a feature typical of eukaryotic primases. These results shed significant light on the evolutionary pathway of primase, and are consistent with the hypothesis of the Asgard origin of Eukarya. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵# Co-first Author