Insight into the Human DNA Primase Interaction with Template-Primer

Abstract

DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58 C ) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58 C . The specific mode of interaction with a template-primer involving the terminal 5-triphosphate of RNA and the 3-overhang of DNA results in a stable complex between p58 C and the DNA/RNA duplex. Our results explain how p58 C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58 C . These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.The four-subunit primase-polymerase ␣ (Prim-Pol␣) 3 complex possessing DNA primase and DNA polymerase active sites is important for genome replication in eukaryotes (1, 2). PrimPol␣ synthesizes the chimeric RNA-DNA primers for replicative DNA polymerases ⑀ and ␦ (3, 4). In humans, the primase heterodimer contains a small catalytic subunit (p49; also known as p48, PRIM1, Pri1, and PriS) and a large regulatory subunit (p58; also known as PRIM2, Pri2, and PriL). Pol␣ is composed of a large catalytic subunit (p180) and a small accessory subunit (p70). p58 and p70 are connected with p180 through the interaction with a small C-terminal domain (p180 C ) that defines the tight coordination of the RNA-and DNA-polymerizing activities (5-7).Eukaryotic primases have a minimal specific recognition site on DNA and only require a pyrimidine to template the 5Ј-terminal nucleotide of the primer (8, 9). RNA primer synthesis begins with a rate-limiting initiation step that includes binding of the DNA template and two NTPs followed by dinucleotide synthesis (10). Subsequently, primase elongates the generated dinucleotide to the unit-length primer (8 -10-mer) and terminates synthesis. This unique counting ability of DNA primases, which results in RNA primers that are optimal for extension by Pol␣, has a complex mechanism that is currently unclear. Recent structural data revealed that the primase active site located on p49 uses the common mechanism of nucleic acids synthesis, where the catalytic aspartates coordinate two divalent ions and the triphosphate moiety of the incoming NTP (11,12).The large subunit of human primase is composed of two separate domains connected with a long linker, which indicates a significant conformational flexibility of this subunit (13). The N-terminal domain (p58 N ) provides a platform for interactions with p49 and P...

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

“…Furthermore, the electrostatic interactions are extended by the DNA backbone of the DNA/RNA duplex. This structure explains why the elimination of 5Ј-triphosphate and/or 3Ј-overhang abolished the interaction of DNA/RNA with p58 C as well as with fulllength primase in our recent study (26).…”

“…However, the mechanism of switch and involved domains remained unknown. Recently we found that p58 C makes a tight complex with a 7-bp DNA/RNA duplex with almost the same affinity for the template/primer as a whole primase (26). Moreover, the final and intermediate products of primase reaction have similar affinity for primase (Fig.…”

“…We recently defined the elements of the DNA template and the RNA primer that are important for interaction with the human primase and found that all of them interact with p58 C (26). These findings allowed us to design the optimal substrate and prepare a stable p58 C -D/R complex for crystallographic studies.…”

“…The p58 C -D/R structure, elongation complex models, and biochemical data (24,26,42) show that p58 C has a much stronger role in template/primer recognition than the catalytic p49 subunit. This is consistent with a distributive nature of RNA synthesis pointing to the high probability of p49 dissociation from the template/primer after incorporation of each nucleotide (Fig.…”

“…Oligonucleotides without 5Ј-triphosphate were obtained from IDT Inc. RNA primers containing the 5Ј-triphosphate (P1-P4) were obtained as described previously (26). DNA duplex T9-P5 and RNA polymerase of bacteriophage T7 were used for synthesis of the ribo-primer P1 (5Ј-pppGGCGGC).…”