Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA

Abstract

The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication. However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the single-stranded DNA or by a direct physical interaction between the DnaB and the DnaG proteins. In this paper we present evidence supporting direct interaction between the two proteins. We have mapped the surfaces of interaction on both DnaG and DnaB and show further that mutations that reduce the physical interaction also cause a significant reduction in primer synthesis. Thus, the physical interaction reported here appears to be physiologically significant.The multiprotein complex that initiates, elongates, and terminates DNA replication has been likened to a protein machine (1). The various component proteins of this machine have to work coordinately to successfully carry out the complex task of DNA replication. Thus, careful analyses of the interactions amongst the replisomal proteins are of critical importance for gaining further insights into the mechanism and control of the three steps of DNA replication. In both Escherichia coli and in mammalian cells, discontinuous synthesis of Okazaki fragments is primed by a class of enzymes called primases (2) that move along the length of the DNA template as a part of a multiprotein primosomal complex, synthesizing RNA primers at intermittent locations on the template DNA (3). Although a great deal of information has been uncovered regarding the enzymology of fork movement in prokaryotes and eukaryotes (3, 4), a number of significant questions still remain unanswered regarding the protein-protein interactions that control and drive fork movement.Studies on priming of phage single-stranded DNA (ssDNA), which is not coated with ssDNA binding protein, have shown that DnaB helicase (5) is needed along with DnaG primase for optimal primer synthesis (3). Two alternative mechanisms have been proposed to account for the need for DnaB in primer synthesis. One hypothesis was that DnaB interacted with ssDNA and created a conformation that allowed DnaG to load on to the DNA. This idea was based on the observation that DnaB binding induced a change in the secondary structure of ssDNA (5). The alternative hypothesis was that DnaB interacted directly with DnaG and with ssDNA, thus facilitating the loading of the primase on the template and͞or the activation of the enzyme (3). Although functional interaction between the C terminus of DnaG with DnaB was reported by Marians and coworkers (7,8), critical evidence showing physical interaction between the two protein has been elusive.To distinguish between the two alternative models of activation of the primase and͞or its loading on DNA, we have investigated the possible physical interaction between the helicase and the primase of Escherichia coli. In this paper we present evidence suppo...

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