Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering

Abstract

Initiation of DNA synthesis in genomic duplication depends on primase, the DNA-dependent RNA polymerase that synthesizes de novo the oligonucleotides that prime DNA replication. Due to the discontinuous nature of DNA replication, primase activity on the lagging strand is required throughout the replication process. In eukaryotic cells, the presence of primase at the replication fork is secured by its physical association with DNA polymerase α (Pol α), which extends the RNA primer with deoxynucleotides. Our knowledge of the mechanism that primes DNA synthesis is very limited, as structural information for the eukaryotic enzyme has proved difficult to obtain. Here, we describe the crystal structure of human primase in heterodimeric form consisting of full-length catalytic subunit and a C-terminally truncated large subunit. We exploit the crystallographic model to define the architecture of its nucleotide elongation site and to show that the small subunit integrates primer initiation and elongation within the same set of functional residues. Furthermore, we define in atomic detail the mode of association of primase to Pol α, the critical interaction that keeps primase tethered to the eukaryotic replisome.

Crystal Structure of the Human Primase

“…This interaction could be important for catalytically productive positioning of the DNA template on p49. Consistent with our model, it has been shown recently that Arg-56 and Tyr-54 are necessary for primase activity (16).…”

“…1C), indicating that p58 N can adopt different conformations. Such flexibility was also observed in the structures of human and Sso primases with a deleted C-terminal domain of the large subunit and can play a role in PrimPol ␣ function (16,26). The proposed NTP/DNA-binding interface of p58 C (17,18) is facing the p49 active site (Fig.…”

“…We designed the template-1 to possess only one pyrimidine (thymine) to allow for a single unique primer start position in comparison with commonly used poly(dT) templates. It was shown recently that p70-p180 C stabilizes activity of the human primase and increases its processivity (5,16). That is why for the activity assays we used primase constructs in complex with p70-p180 C .…”

“…It has also been proposed that the large subunit helps to "count" the product size and participates in translocation of the primertemplate to the Pol ␣ active site (10,12). The N-terminal part of the large subunit (p58 N in human primase) provides a platform for interactions with p49 and Pol ␣ (5,16). Most structural elements contributing to NTP and DNA binding by the large subunit are located in the C-terminal half of the protein (p58 C in human primase) (11,(17)(18)(19)(20).…”

“…These data and mapping of the active site of mouse p49 by site-directed mutagenesis allowed determination of the positions of three invariant catalytic aspartates (27). Recently, the crystal structures of the catalytic subunit of yeast primase and the human p49-p58(1-253) complex have been determined (16,28). These results confirmed the early idea proposing similar organization of the active site in eukaryotic and archaeal primases and the role of three catalytic aspartates in the coordination of two divalent ions and NTP (23,24).…”