Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome

Abstract

The human primosome, a 340-kilodalton complex of primase and DNA polymerase ␣ (Pol␣), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases ␦ and ⑀. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58 C ) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Pol␣, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58 C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.In eukaryotes, the primosome, a tight complex of DNA primase and DNA polymerase ␣ (Pol␣), 4 synthesizes primers for both leading and lagging strands in a highly coordinated fashion (1, 2). The primosome is indispensable for initiation of replication and has a large impact on genome stability (3-6). RNA primer synthesis by primase involves three steps: initiation, elongation, and termination (7,8). During the rate-limiting initiation step, primase binds the DNA template and two ribonucleotide triphosphates (NTPs) and catalyzes the formation of a dinucleotide (9, 10). Further synthesis of the RNA primer is much faster but restricted, because of the intrinsic property of primases to count the primer length and terminate synthesis after incorporation of 8 -10 nucleotides (7). Next, the mature so-called "unit length" RNA primer is intramolecularly translocated to Pol␣ for the subsequent extension by dNTPs, and the primase became inhibited by an unknown mechanism (9,11,12). Orchestration of all these steps requires changes in primosome conformation (13).Human Pol␣ (Fig. 1A) is comprised of a large catalytic subunit (p180) and a smaller accessory subunit (p70), connected by the C-terminal domain of p180 (p180 C ) containing two conserved zinc-binding modules, Zn1 and Zn2 (14 -16). p70 consists of an N-terminal (p70 N ), a phosphodiesterase, and oligonucleotide/oligosaccharide-binding (OB) domains (14, 17). The globular p70 N is attached to the phosphodiesterase via a flexible linker (amino acid residues 79 -156) (14, 18) and participates in interactions with other DNA replication proteins (19). The catalytic core of p180 (p180core) and p180 C -p70 are connected by a 15-residue linker (1251-1265) (13). Human primase consists of catalytic (p49) and regulatory (p58) subunits (20). p58 has two distinct domains, N-terminal (p58 N ) and C-terminal (p58 C ), connected with an 18-residue linker (253-270) (21). p58 N interacts with p49 and connects primase with Pol␣ (22, 23), and an iron-sulfur cluster containing p58 C plays an important role in substrate binding and primase acti...

Comment on “The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport”

“…Both the 5′-triphosphate of a primer and the 3′-overhang of a template are critical for DNA:RNA binding by p58C and primase (4, 6) (Fig. 1C, highlighted in blue).…”

“…In the conserved p58C/substrate complex structure Y345 interacts with the γ-phosphate of the initiating NTP (Fig. 1B) (4), which is rather sensitive to the binding environment (8,9). For example, mutation of Arg306 analog in yeast primase, which makes two contacts with initiating NTP (4), completely abrogates de novo RNA synthesis (9).…”

Proficient Replication of the Yeast Genome by a Viral DNA Polymerase

“…tured NTD and the exonuclease and polymerase domains, but lacks the unstructured N-terminal tail and its CTD. The CTD of Pol ␣ serves as a structural model for this domain in the other B-family DNA polymerases (19,20).…”

“…However, biochemical studies of Pol ␦ and Pol have shown that the C-terminal 4-cysteine lobe ligands an iron-sulfur cluster in the 2ϩ coordination state (21,22). The CTDs of Pol ␣, Pol ␦, and Pol ⑀ each bind a distinct B subunit, called Pol12, Pol31, and Dpb2, respectively, in budding yeast (19,21,23), and these B subunits show both sequence and structural conservation (19,20,24,25). Pol has appropriated the B subunit from Pol ␦ to elaborate its own four-subunit assembly (Rev3-Rev7-Pol31-Pol32) (22, 26 -28).…”

“…The CTD sequences of the four eukaryotic enzymes are highly conserved, suggesting a common three-dimensional structure of the CTD. Only the structure of the CTD of Pol ␣ has been determined (19,20). It shows an elongated, bilobal form, in which the two lobes are connected by a three-helical bundle.…”