Identification of a Region of Escherichia coli DnaB Required for Functional Interaction with DnaG at the Replication Fork

Pearl Chang,Kenneth J. Marians

doi:10.1074/jbc.m001800200

Abstract

The fundamental activities of the replicative primosomes of Escherichia coli are provided by DnaB, the replication fork DNA helicase, and DnaG, the Okazaki fragment primase. As we have demonstrated previously, DnaG is recruited to the replication fork via a transient protein-protein interaction with DnaB. Here, using site-directed amino acid mutagenesis, we have defined the region on DnaB required for this protein-protein interaction. Mutations in this region of DnaB affect the DnaB-DnaG interaction during both general priming-directed and phiX174 complementary strand DNA synthesis, as well as at replication forks reconstituted in rolling circle DNA replication reactions. The behavior of the purified mutant DnaB proteins in the various replication systems suggests that access to the DnaG binding pocket on DnaB may be restricted at the replication fork.

Highlights

In bacteria, the DNA unwinding and Okazaki fragmentpriming functions at the replication fork are provided by a primosome, a multienzyme conglomerate that moves processively along the lagging-strand template [1]
Initial studies demonstrated that DnaG, which had been identified as a primase [10, 11], was not present in restart primosomes formed in the absence of DNA synthesis and isolated by gel filtration bound to ␾X174 ss(c)DNA
Because in the rolling circle system the nascent leading strand is the lagging-strand template, Okazaki fragment size can be made to vary at a fixed concentration of DnaG by altering the rate at which the lagging-strand template is generated, i.e. by altering the rate of DnaB-catalyzed unwinding at the replication fork. We considered this explanation unlikely because in this scenario DnaB Y105A would have to have at least a 15-fold greater rate of DNA unwinding at the replication fork than the wild-type protein, we compared the rate of replication fork progression for the wild type and mutant proteins directly

Summary

Introduction

The DNA unwinding and Okazaki fragmentpriming functions at the replication fork are provided by a primosome, a multienzyme conglomerate that moves processively along the lagging-strand template [1]. The replicative primosome formed at oriC requires DnaB, DnaC, and DnaG for assembly, whereas the replication restart primosome (formerly the ␾X174-type primosome [3]), which can form at D loops [4], requires PriA, PriB, DnaT, and possibly PriC, in addition to the former three proteins, for assembly [3] Primosomes provide both the DNA unwinding and Okazaki fragment-priming functions of the replisome. During replication fork reactivation, PriA identifies the site for restart primosome loading [2, 3, 8], and it is probably a protein-protein interaction between DnaT and DnaB that mediates transfer of DnaB to SSB-coated DNA [9]. At identical concentrations, DnaG Q576A directs the synthesis of Okazaki fragments that are at least 15-fold longer in size than those directed by the wild type protein [18]

Methods

Results

Conclusion