Crystal structure of the C-terminal domain of human DNA primase large subunit: implications for the mechanism of the primase-polymerase α switch

Abstract

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes' large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.

Figure 1

A stereoview of the cartoon representation of human p58C/3Q36. the 4Fe-4S cluster and the side chains of coordinated cysteine residues are shown as sticks.

Figure 2

Comparison of the human p58C/3Q36 structure determined in current work with the corresponding domain structures reported for the yeast and human proteins. (A) Structure-based amino acid sequence alignment of the human and yeast primase 4Fe-4S domains. The sequences corresponding to α-helices and β-strands are highlighted by grey and yellow color, respectively. Conserved residues are shown in red and residues not in the final model are highlighted by cyan color. Residues involved in 4Fe-4S cluster coordination are indicated by triangles. (B and C) Comparison of the human p58C/3Q36 with the structures of the (B) yeast (3LGB) and (B) human (3L9Q) 4Fe-4S cluster domains. The labeled regions exhibiting the significant differences are discussed in the text

Figure 3

Examples of Far UV spectra of human p58C/3Q36 obtained at lower and high pH. The spectra are shown for the protein dialyzed against MES pH 6.5 and 40 mM NaCl (smooth line), and bicine pH 9.0 and 200 mM Li₂SO₄ (dotted line).

Figure 4

Crystal packing of the primase large subunit 4Fe-4S cluster domains. The figure highlights surrounding of helices α4, α5 and α6 in (A) human p58C/3Q36 and (B) yeast PriL-CTD and (C) β-strands in human p58C/3L9Q.

Figure 5

Comparison of yeast PriL-CTD and human p58C/3Q36 with the ssDNA-binding fragment of DASH cryptochrome 3 from Arabidopsis thaliana (PDB code 2VTB). The yeast PriL-CTD (residues 334–423) and human p58C/3Q36 (285–374) were superimposed with A. thaliana DASH cryptochrome 3 (373–457) with rmsd of 1.36 Å for 72 matched C_α atoms and rmsd of 1.32 Å for 66 matched C_α atoms, respectively. The protein fragments are drawn as cartoons. The FAD and ssDNA bound to a DASH cryptochrome 3 are shown as sticks.

Figure 6

A proposed model of primer length counting and primer-template dissociation from DNA primase. (A) Initiation of primer synthesis. (B) Synthesis of unit length RNA primer causes a sterical hindrance. (C) Sterical hindrance induces the conformational change, resulting in RNA primer-template dissociation from primase and its transfer to Pol α.