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.

Fig. 1.

Structure of the human primase, comprising full-length PriS and a truncated version of PriL, lacking its C-terminal Fe-S domain. Front and top views of the primase are shown. The primase is drawn as a ribbon, colored orange for PriS and blue for PriL. The position of the zinc atom in PriS is also shown. The position of the active site in PriS is indicated by a black arrow.

Fig. 2.

The prim fold of human primase. Only secondary structure elements that make up the prim fold are shown, as blue ribbons for α-helices and light green arrows for β-strands. The small arrows indicate the points of insertion of the smaller, helical domain (omitted from the figure for clarity). The position of the active site in PriS is indicated by a black arrow.

Fig. 3.

Functional analysis of active-site PriS residues by structure-based alanine mutagenesis. A and B show the result of the primase initiation and elongation assays, respectively. The initiation assay measures the ability of primase to initiate primer synthesis in the presence of single-strand poly(dT) DNA and ATP. In the elongation assay, an oligo(A) primer is present in the reaction, in addition to poly(dT) DNA and ATP; the assay measures the ability of primase to initiate primer synthesis and/or extend an existing RNA primer. Briefly, reaction products were separated by denaturing electrophoresis on polyacrylamide gels and analyzed by phosphorimaging. For each alanine mutant, two different primase concentrations were tested in the initiation assay. The R306A mutant refers to the residue in the PriL-CTD. See Methods for details.

Fig. 4.

The nucleotide elongation site of human primase. (A) Detailed view of PriS with bound UTP. PriS is drawn as a thin tube, with secondary structure elements colored cyan for α-helices and light green for β-strands. The side chains of residues assayed for primer synthesis in the functional assays are shown as stick models. The UTP is drawn as a ball-and-stick model. Carbon atoms are colored white, oxygens red, nitrogens blue, phosphates orange, and sulfurs yellow. The hydrogen-bonding interactions between UTP and the side chains of H166 and H315, as well as the main-chain atoms of L316 and K318 are shown as pink lines. The position of the two magnesium ions in the active site, as well as that of the zinc atom in the adjacent Zn-binding motif, is shown as light gray and dark gray spheres, respectively. (B) A cartoon of primase during the initiation (Left) and the elongation step (Right). The ovals in PriS are labeled “I” and “E” to indicate the initiation and elongation NTP-binding sites in PriS, respectively. The result of the functional assays support a mechanism of RNA primer synthesis where the initiation site is occupied by the 3′-end of the primer during elongation.

Fig. 5.

Complex of human primase bound to the primase-binding motif of Pol α. (A) Pol α binds to the PriL subunit of primase. PriS and PriL are drawn as ribbons, colored in orange and blue, respectively. The primase-binding motif of Pol α is drawn as a yellow tube. A cartoon of Pol α/primase shows tethering of primase to the replisome by the interaction described in the panel. (B) Detailed view of the Pol α–primase interface. PriL and Pol α are depicted as thin tubes, in medium blue and yellow, respectively; the α-helices in PriLα are rendered as cylinders. Main-chain and side-chain atoms of selected residues at the interface between PriL and Pol α are drawn as sticks, with red oxygens, blue nitrogens, and carbons in light blue and pale yellow for PriL and Pol α, respectively. Hydrogen bonds are shown as pink lines. α-Helices in PriL are labeled as in Fig. S4.