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, 290 (9), 5635-46

Crystal Structure of the Human Primase

Affiliations

Crystal Structure of the Human Primase

Andrey G Baranovskiy et al. J Biol Chem.

Abstract

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme's conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.

Keywords: Crystal Structure; DNA Primase; DNA Replication; Iron-Sulfur Protein; Protein Complex; Zinc.

Figures

FIGURE 1.
FIGURE 1.
Overall structure of the human primase. A, schematic representation of the domain organization. The orange lines and red lines in the schematics present relative positions of the residues coordinating zinc (Cys-121, Cys-122, Cys-128, and Cys-131) and the 4Fe-4S cluster (Cys-287, Cys-367, Cys-384, and Cys-424), respectively. B, schematic representation of p49-p58. The p49, p58N, p58C, and the linker between p58N and p58C are colored cyan, green, light pink, and gray, respectively. Zinc is shown as an orange sphere, and the 4Fe-4S cluster is shown as a space-filled representation, with iron and sulfur atoms colored blue and yellow, respectively. The disordered regions in p49 are shown by dashed lines. Side chains of catalytic aspartates on p49 and residues forming the proposed NTP/DNA binding site on p58C are shown as sticks and colored magenta. C, comparison of the two primase molecules in an asymmetric unit by superposition of p49. The p58N domain from one molecule (with p58C) is colored green, and that from a second molecule (without p58C) is colored red. p49 and p58C are omitted for clarity. The α-helices responsible for conformational flexibility of p58N are labeled.
FIGURE 2.
FIGURE 2.
Schematic representation of the p49 and p58 subunits with numbering of secondary structure elements. Both subunits are colored by a gradient from blue (N terminus) to red (C terminus). Zinc is shown as an orange sphere; the iron-sulfur cluster is omitted for clarity. Disordered regions in p49 are shown by dashed lines.
FIGURE 3.
FIGURE 3.
Alignment of the human and Sso primase molecules. The catalytic (PriS) and large (PriL) subunits of Sso primase are colored salmon and gray, respectively. Zinc in PriS is shown as a magenta sphere.
FIGURE 4.
FIGURE 4.
Structural comparison of catalytic subunits from the human and archaeal primases. A–E, schematic representation of catalytic subunits from human and archaeal primases. The catalytic aspartates in active sites are highlighted as balls and sticks. The oxygen is colored red, and zinc is shown as an orange sphere. F, superimposition of catalytic aspartates from the structures of archaeal and human primases. Aspartates are depicted by a stick representation, and carbons are colored cyan (human), light green (Pho; PDB code 1V33), dark green (Pfu; PDB code 1G71), salmon (Sso; PDB code 1ZT2), and gray (Sis; PDB code 3M1M). The p49 active site is represented as a schematic where the secondary structure elements are shown with 50% transparency. The positions of catalytic aspartates in p49 are labeled.
FIGURE 5.
FIGURE 5.
Close-up view of the p58N-p58C interaction interface. The protein is represented as a schematic and colored according to Fig. 1B. Side or main chains making the hydrogen bonds between p58N and p58C are shown as sticks. Hydrogen bonds are drawn with red dashed lines.
FIGURE 6.
FIGURE 6.
Close-up stereo view of the hydrophobic interface between p49 and p58. Side chains of the residues involved in hydrophobic interactions between the two subunits are shown as sticks. Each subunit is colored according to Fig. 1B. The secondary structure elements are shown with 50% transparency.
FIGURE 7.
FIGURE 7.
Close-up stereo view of the hydrophilic contacts at p49-p58 interface. Side or main chains making the hydrogen bonds between two subunits are shown as sticks. Water molecules involved in intersubunit interactions are depicted by red spheres. Each subunit is colored according to Fig. 1B. The secondary structure elements are shown with 40% transparency.
FIGURE 8.
FIGURE 8.
Conserved main-chain to main-chain contacts between catalytic and large subunits of the human (A) and Sso (B) primases. The color scheme is the same as in Fig. 3. Main chains of the residues involved in intersubunit interactions are shown as sticks. The hydrogen bonds are depicted by blue dashed lines.
FIGURE 9.
FIGURE 9.
Activity analysis of the human primase and its deletion mutants. A, de novo activity on template-1 (5′-A15TA7). p58(266–456) was added at a 2-fold molar excess over p49-p58(1–265). B, de novo activity of the wild-type primase on template-2 (5′-AAA(GA)6TA7). C, extension assay using fluorophore-labeled RNA primer annealed to template-3. Lane 1, control incubation (no enzyme); lanes 2 and 7, wild-type primase; lanes 3 and 8, Prim-Δ5; lanes 4 and 9, Prim-Δ15; lanes 5 and 10, Prim-Ins5; lanes 6 and 11, p49-p58(1–265). Reactions were incubated for 1 min (lanes 2–6) and 10 min (lanes 1 and 7–11) at 35 °C. The activity of primase samples was analyzed in the presence of p70-p180C. Only the 5′-end of RNA products was labeled because of using [γ-33P]ATP (A and B). D, analysis of the purity of the human primase samples. Lane 1, EZ-Run Rec protein ladder (Fisher); lane 2, Prim; lane 3, Prim-Δ5; lane 4, Prim-Δ15; lane 5, Prim-Ins5; lane 6, p49-p58(1–265); lane 7, p58(266–456). Samples were run on 10% SDS-PAGE, and proteins were detected by Coomassie Blue staining.
FIGURE 10.
FIGURE 10.
Modeling of Prim-DNA/RNA-CTP ternary complex. A, overall view of the ternary complex. The color scheme is the same as in Fig. 1. B, close-up view of the p49-DNA/RNA-CTP complex. The p49 surface is represented by the vacuum electrostatic potential. The carbons of CTP, DNA, and RNA are colored gray, green, and cyan, respectively. C, close-up view of the p49-DNA interaction interface. Carbons of Tyr-54, Arg-56, Lys-311, and Asn-314 are colored brown. The potential hydrogen bonds between p49 and DNA are shown by pink dashed lines.

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