Primases are classified into two major groups first the

Primases are classified into two major groups: first, the DnaG primases, found in bacteria and bacteriophages, and second, the archaeoeukaryotic primases. Remarkably, bacterial and archaeoeukaryotic primases have no structural similarity and, presumably, evolved independently (Leipe et al., 1999). The archaeoeukaryotic primases are found in eukaryotes and archaea as well as in diverse mobile genetic elements. Cellular archaeoeukaryotic primases are usually heterodimeric with a small catalytic subunit (PriS, cd04860) and a large accessory subunit (PriL, cd06560). Additionally, the primase heterodimer may be part of a larger complex; e.g., in eukaryotes with polymerase α. In nearly all cases, the conserved domain of the small catalytic subunit and the conserved domain of the large subunit are encoded on two genes. Gene fusions of cellular archaeoeukaryotic primases are only rarely reported (most notable Nanoarchaeum equitans [NEQ395]; Makarova and Koonin, 2013). PriS adopts a fold related to RRMs (RNA recognition motifs), with a four-stranded β sheet and two α helices (Iyer et al., 2005). In all enzymatic studies, PriS alone is unable to synthesize a primer despite bearing the catalytic residues and forming the active site. Thus, it appears that it requires the help of another domain to synthesize a primer, possibly the PriL subunit. The C-terminal domain of PriL (PriL-CTD) is largely helical and embeds an iron sulfur cluster (Sauguet et al., 2010). Based on the structural resemblance between PriL-CTD and cryptochromes, it was suggested that the ssDNA path found in the cryptochrome may represent the binding site of the DNA template of the primase and the flavin boc boc dinucleotide (FAD) binding site in the cryptochrome, the position of the initiating and/or elongating nucleotide (Sauguet et al., 2010). Thus, it was hypothesized that PriL could contribute to primer synthesis by binding the template DNA and/or the nucleotides. Indeed, it could be shown that yeast PriL-CTD binds, albeit weakly, single- and double-stranded DNA (dsDNA) with dissociation constants of 70 μM and 180 μM, respectively (Sauguet et al., 2010), and that human PriL also binds ssDNA and dsDNA with a KD of ∼1 μM (Vaithiyalingam et al., 2010). More recently, it was demonstrated that the affinity of the human PriL-CTD toward a primer-template DNA with a 3′ and a 5′ template overhang and 5′ terminal triphosphate at the primer end is even bound with a KD of ∼30 nM. Binding affinity is strongly reduced in the absence of the 3′ overhang or the 5′ terminal triphosphate; thus, PriL-CTD appears to bind especially well to the 5′ end of a native primer (Baranovskiy et al., 2015). Consequently, it has been proposed that the preparation of dinucleotide synthesis may take place at the interface of PriS and PriL-CTD and that PriL-CTD may bind template and/or nucleotides prior to the catalytic steps. Apart from the cellular primases, archaeoeukaryotic primases are also present in mobile genetic elements. Here the conserved domain PriS is often associated with conserved domains that are less well characterized, in particular PriCT_1 (cl07362) and PriCT_2 (cl07361). A recent study highlights that there is a structural resemblance between PriCT-1, PriCT-2, PriL-CTD, and PriX (Kazlauskas et al., 2018), with PriX being a recently discovered additional subunit of primases in Crenarchaeota (Holzer et al., 2017, Liu et al., 2015). This raises the question whether all of these rather small helical domains associated with PriS contribute in a similar way to primer synthesis. In our group, we have investigated the primase part of the multifunctional replication protein from the archaeal plasmid pRN1 (Figure 1A). In contrast to other archaeoeukaryotic primases, this enzyme is template-specific because priming only occurs at a GTG motif (Beck et al., 2010; Figure 1B). In addition, it synthesizes a mixed primer, with the first base (i.e., 5′) being exclusively a ribonucleotide and the remaining ones being exclusively deoxynucleotides (Beck and Lipps, 2007). The primase part of the replication protein is further divided into two domains, the catalytic Prim_Pol domain (cd04859) and the pRN1_helical domain (pfam13010, named here helix bundle domain [HBD]). The Prim_Pol domain as well as the HBD are structurally related to the cellular archaeoeukaryotic counterparts PriS and PriL-CTD, respectively (Boudet et al., 2015). The crystal structures of the catalytic Prim_Pol domain alone (Lipps et al., 2004) and of the free functional pRN1 primase encompassing both Prim_Pol and HBD (Beck et al., 2010) have been solved, but crystals with bound substrates could never be obtained. We therefore investigated the structural features of this enzyme with nuclear magnetic resonance (NMR) spectroscopy.