br Introduction Primases have an important function in DNA r

Introduction Primases have an important function in DNA replication. They synthesize de novo, on single-stranded DNA (ssDNA), a primer that is then extended by DNA polymerases. In the context of the replisome, primer synthesis is repeatedly required on the lagging strand. Although DNA primases are among the most error-prone polymerases, the integrity of the newly synthesized DNA is efficiently preserved. Primase-generated RNA stretches are degraded during Okazaki fragment maturation; DNA polymerases fill the gaps, and DNA ligases seal the remaining nicks (Arezi and Kuchta, 2000, Frick and Richardson, 1999, Griep, 1995, Kuchta and Stengel, 2010). Primer synthesis can be divided into two fundamental phases. The first and probably rate-limiting step is the formation of a first phosphodiester bond between the two first nucleotides, which requires hydrolysis of the triphosphate of the elongating nucleotide, whereas the triphosphate of the initiating nucleotide becomes the 5′ end of the primer (Frick and Richardson, 2001). The second phase of primer synthesis is repeated NMS-1286937 receptor of the primer by addition of ribonucleotides at the 3′ hydroxyl group until a defined primer length is reached. This phase is very similar to the chain elongation reaction of DNA polymerases, and the presence of acidic catalytic residues suggests that the extension reaction is catalyzed by the two-metal-ion mechanism of DNA polymerases (Augustin et al., 2001, Keck et al., 2000, Steitz et al., 1994). Although the chemistry of dinucleotide synthesis and primer elongation is similar and likely involves the same catalytic residues, major questions remain, in particular how the primases are able to simultaneously bind and position the three substrates (template, initiating nucleotide, and elongating nucleotide) required for dinucleotide formation and how the primase terminates primer synthesis. 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, Sex chromosome 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).