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 (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C. 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 p58C and the DNA/RNA duplex. Our results explain how p58C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.

Highlights

  • 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]

  • Triphosphate and the 3Ј-Overhang of DNA/RNA Duplex Are Important for Interaction with Primase—RNA primers synthesized by primases or RNA polymerases contain the triphosphate moiety at the 5Ј-terminus (TriP) [23, 24]

  • To examine the role of the TriP in the interaction of the human primase with the substrate, we used the DNA template T1, which could be annealed with a 7-mer RNA primer with or without TriP (P1 and P2, correspondingly) and has the 5Ј- and 3Ј-overhangs to mimic the natural primase substrate

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Summary

Introduction

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]. TriP and the 3Ј-Overhang Binding Sites Are Located on p58C—Several pieces of evidence obtained earlier indicate that p58C somehow participates in the interaction with substrates because it plays an important role in primase function, especially during dinucleotide synthesis (8, 16, 17, 26 –28).

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