Identification of a region of Escherichia coli DnaB required for functional interaction with DnaG at the replication fork

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.