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-Å 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.he replisome is a dynamic assembly of proteins that carries out eukaryotic DNA replication. The first step in the initiation of DNA replication on both the leading and lagging strand ssDNA is the synthesis of a short (8-10 nucleotide) RNA primer by a polymerase termed a DNA primase (1-3). DNA primases are unique because they are the sole polymerases capable of de novo synthesis on a ssDNA template. Although integral to the replisome, the activity of DNA primase is not limited to DNA replication; primase is also an essential component of the DNA damage response and plays a role in telomere maintenance (4, 5).Although all primases serve a common function, their architectures and modes of interaction with other protein factors vary among species (6, 7). The human DNA primase is part of the heterotetrameric polymerase α-primase (pol-prim) complex, which generates short (∼30 nucleotide) stretches of DNA primed with chimeric RNA-DNA hybrids that are handed off to the primary replicative DNA polymerases. The p48 and p58 subunits of human pol-prim form the DNA primase heterodimer. Although the p48 subunit contains the RNA polymerase catalytic active site, both subunits are required for synthesis of short RNA primers (8, 9). The p58 subunit is proposed to play various roles, including stabilizing the p48 subunit, recognizing the DNA template, and promoting initiation and elongation of primers (9, 10).Considerable knowledge about the molecular mechanism of DNA priming has been obtained from structural studies of archaeal and bacterial primases (11, 12). In contrast, the mechanism of DNA priming ...

Crystal Structure of the Human Primase

“…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. 1B), which may facilitate their cooperation during RNA synthesis in the cis-position (see "Discussion").…”

“…C-terminal domain of p58 binds single-and double-stranded DNA with micromolar affinity (18). Photocross-linking of single-stranded DNA with human primase revealed that only p58 reacted with DNA upon photolysis (12).…”

“…C-terminal domain of the large subunit of the yeast and human primases revealed an iron-sulfur cluster (4Fe-4S) coordinated by four cysteines (17,18,21). The results of structural studies confirmed previously accumulated genetic and biochemical data on the role of these conserved cysteine residues in the structural stability and activity of primase (14,19,20,22).…”

“…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). The crystal structures of the * This work was supported, in whole or in part, by National Institutes of Health (NIH), NIGMS, Grant GM101167 (to T. H. T.) and NIH, NCI, Grant CA129925 (to Y. I. P.).…”

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

“…Although the primary active site for RNA primer synthesis resides in the PriS subunit, PriL is important for regulation of primase activity and primer transfer to Pol α (5-8). Further insight into PriL function was provided by the observation that a conserved C-terminal Fe-S domain of PriL, PriL-CTD, is essential for initiation of primer synthesis, but dispensable for its subsequent elongation (9)(10)(11)(12)(13).…”