Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase

Abstract

DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-A resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.

Fig. 1.

The three-dimensional structure of p58C. Helices and β-strands are colored cyan and blue, respectively. The 4Fe-4S cluster and the side chains of key residues Arg306 and Lys314 are shown in ball and stick representation.

Fig. 2.

Binding of DNA by p58C. (A) Molecular surface of p58C colored on the basis of electrostatic field potential (red, -7 k_BT; blue +7 k_BT) with the positions of Arg302, Lys314, and Trp327 highlighted. (B) Plots of increase in fluorescence anisotropy as a function of p58C concentration for labeled 12-mer ssDNA (open black circles), 12-mer dsDNA (closed blue squares), and ss/dsDNA junction with 12 base-pair duplex (closed red circles).

Fig. 3.

Interaction of p58C with RPA32C. (A) Isothermal titration calorimetry binding isotherm for titration of RPA32C into p58C showing the raw heat release (Upper) and the integrated heat release (Lower). (B) Binding of p58C to RPA32C monitored by ¹⁵N-¹H heteronuclear single quantum coherence NMR spectra of ¹⁵N-RPA32C in the absence (black) and presence of 1∶0.25 (yellow), 1∶0.5 (cyan), 1∶1 (green) and 1∶2 (red) molar ratios of p58C. (C) Molecular surface of RPA32C colored according to electrostatic potential (red, -7 k_BT; blue +7 k_BT) with key residues labeled.

Fig. 4.

Model of the p58C–RPA32C complex. (A) Ribbon diagram with p58C and RPA32C colored cyan and gold, respectively. Side chains at the interface are shown in ball and stick representation. (B) The same view of A, with RPA32C shown as a molecular surface colored according to electrostatic potential (red, -7 k_BT; blue +7 k_BT). (C) Isothermal titration calorimetry binding isotherm for titration of RPA32C into p58C mutants His299Asp (Left) and Arg302Glu (Right). Upper and lower panels show the raw heat release and the integrated heat release, respectively. (D) Close-up view of the p58C–RPA32C protein interface.