Regulation of bacterial priming and daughter strand synthesis through helicase-primase interactions

Abstract

The replisome is a multi-component molecular machine responsible for rapidly and accurately copying the genome of an organism. A central member of the bacterial replisome is DnaB, the replicative helicase, which separates the parental duplex to provide templates for newly synthesized daughter strands. A unique RNA polymerase, the DnaG primase, associates with DnaB to repeatedly initiate thousands of Okazaki fragments per replication cycle on the lagging strand. A number of studies have shown that the stability and frequency of the interaction between DnaG and DnaB determines Okazaki fragment length. More recent work indicates that each DnaB hexamer associates with multiple DnaG molecules and that these primases can coordinate with one another to regulate their activities at a replication fork. Together, disparate lines of evidence are beginning to suggest that Okazaki fragment initiation may be controlled in part by crosstalk between multiple primases bound to the helicase.

Figure 1

Domain organization of DnaB/DnaG-type helicase/primase systems. Bacterial DnaB is a two-domain protein with an N-terminal interaction domain and a C-terminal helicase domain. DnaG is a three-domain protein with an N-terminal regulatory Zinc Binding Domain, central RNA Polymerase Domain and C-terminal Interaction Domain. Known areas of interaction between the two proteins are shown as arrows. The phage T4 gp41 helicase and gp61 primase are minimalized versions of DnaB and DnaG, respectively. The phage T7 gp4 protein, on the other hand, fuses DnaB- and DnaG-related domains into a single polypeptide.

Figure 2

Structures of the DnaG primase and DnaB helicase. (A) Atomic structures of domains from DnaG (PDB codes 1D0Q, 1DDE, and 1T3W) (21,24,27) are colored as in Figure 1 and connections between domains are shown as dotted lines. (B) The N-terminal domain of DnaB has been solved by NMR and X-ray crystallography (PDB codes 1JWE and 1B79) (20,22), whereas the C-terminal region likely resembles that of the related T7gp4 helicase domain (PDB code 1E0J) (58). Domains are colored as in Figure 1 and for clarity the N-terminal domain has been omitted from the face-on view. Only one of six N-terminal domains is shown in the side view, and the two domains are shown separated from each other. Interestingly, the helical bundles of the N-terminus of DnaB and C-terminus of DnaG adopt very similar folds (21,32). (C) The crystal structure of the T7gp4 primase–helicase (lacking the N-terminal Zinc Binding Domain) illustrates the relative locations of the primase (orange) and helicase (red) modules (12). The linkers between the two domains are shown as black ribbons.

Figure 3

Model for the functional coordination of multiple primases bound to the replicative helicase. Domains of the DnaG primase are outlined in black, while those of the DnaB helicase are not. Domains are colored as in Figure 1. (A) As the helicase unwinds the parental strands, single-stranded DNA containing a trinucleotide primer initiation site (white rectangle) is extruded from the helicase ring and non-specifically passes through the catalytic center of a primase's RNA Polymerase Domain. (B) The Zinc Binding Domain of one primase molecule may form a ternary complex with a second primase's RNA Polymerase Domain and the DNA template, forming an active primase unit to allow the recognition of the primer initiation site and the activation of primer synthesis.