The molecular details of the bacterial helicase-primase complex

DNA replication is one of the most fundamental functions of all living organisms. Understanding the basic mechanisms of action of bacterial DNA replication will be essential not only to extrapolate our findings to the more complex eukaryotic organisms, but also to design new antibacterials in our fight against antibiotic resistance. The helicase-primase complex is a ubiquitous and essential bacterial complex. Our current understanding of the molecular basis of its functions is poorly understood. Recently we have solved the structure of the helicase-interacting domain of the B. stearothermophilus primase protein and we discovered that this domain is structurally homologous to the N-terminal domain of the helicase itself. This was a surprising discovery and one that led us to propose a model to try and explain how the bacterial helicase-primase complex functions. We are now seeking funding to test directly the validity of this model with a series of biochemical experiments. The results of this research will enable us to understand the molecular details of this important complex and will pave the way for the design of new antibacterial drugs that will target this complex and thus bacterial DNA replication.

We have recently solved the NMR structure of the C-terminal DnaB-interacting domain of DnaG. This domain (known as P16) is sufficient to elicit all the functional effects on DnaB that the full-length DnaG can. The structure revealed that P16 consists of two sub-domains (i) the C1 N-terminal subdomain that constitutes a six-helical bundle and (ii) the C2 C-terminal sub-domain that constitutes a helical hair-pin. The C2 sub-domain mediates structurally the interaction with the helicase but the C1 sub-domain is essential for the functional activation of the helicase. A unique and surprising finding was that the C1 sub-domain is structurally homologous to the N-terminal domain (P17) of the DnaB helicase. The structural homology of the C1 subdomain of P16 with the N-domain of the helicase DnaB suggested a structural/functional model for the actual molecular mechanism of action of the helicase-primase complex. We are now seeking funds from BBSRC to test directly this model with a series of mutagenesis experiments and with the construction of chimera proteins. We have carried out structural comparisons between the B. stearothermophilus and E. coli P16 and the P17 domain of the E. coli DnaB and identified a spatially conserved network of residues. These residues must play crucial structural/functional roles in the activities of the helicase-primase complex. We will target all of these residues by mutagenesis to validate their roles. At the same time we will carry our domain swapping experiments between the structurally homologous P16 and P17 domains to reveal whether they are also functional homologues. We have already constructed two chimera proteins and our preliminary data indicate that these domains are indeed functional homologues.