E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

Andrea Bogutzki,Ute Curth,Natalie Naue,Andreas Pich,Lidia Litz

doi:10.1038/s41598-019-51031-0

Abstract

During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

Highlights

The exact replication of the genome is a prerequisite for the division of the bacterial cell
A model has been proposed in which DnaG primase is displaced from the C-terminus of single-stranded DNA-binding protein (SSB) by the χ subunit of the clamp loader in order to allow for the loading of the β2 sliding clamp[13]; it assumes that χ and primase cannot bind to SSB at the same time
Sedimentation velocity experiments showed that the additional presence of rNTPs increases the amount of bound primase significantly, most probably leading to two primases bound per SSB-saturated G4ori

Summary

Introduction

The exact replication of the genome is a prerequisite for the division of the bacterial cell. The tetrameric SSB protein protects the vulnerable single-stranded DNA and holds it in a proper conformation, and engages in several interactions with proteins involved in DNA replication, repair and recombination via its highly conserved amphipathic C-terminus[5] Amongst these proteins are the χ subunit of the clamp loader[6,7,8,9,10,11] and DnaG primase[12,13]. A model has been proposed in which DnaG primase is displaced from the C-terminus of SSB by the χ subunit of the clamp loader in order to allow for the loading of the β2 sliding clamp[13]; it assumes that χ and primase cannot bind to SSB at the same time This model has been controversially discussed, as SSB forms stable tetramers and four C-termini are available at the SSB protein bound next to the primer. Elongation by more than 10 nucleotides, led to the dissociation of one primase molecule, whereas complete conversion of ssM13Gori to its double-stranded form resulted in complete release of primase

Methods

Results

Conclusion