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

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.

Transcriptomic Profile Analysis of Streptococcus mutans Response to Acmella paniculata Flower Extracts

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Distal Mutations in the β-Clamp of DNA Polymerase III* Disrupt DNA Orientation and Affect Exonuclease Activity

“…Helicase (DnaB) is a protein located near the replication fork and is responsible for unwinding the double-stranded DNA (dsDNA) . DNA Polymerase III (Pol III) in complex with the β-clamp replicates DNA in the 5′ to 3′ direction on both the leading and lagging strand with the help of a primase. , Lastly, the ATP-dependent clamp loader occupies a large space within the replisome and consists of 5 subunits: δ A , γ B , γ C , γ D , and δ E . This complex is responsible for opening the β-clamp and positioning it on the DNA.…”