Professor Heath Murray - Newcastle University

Professor Heath Murray

Systematic reduction of a bacterial replication origin reveals the core sequence elements

Bacteria initiate the bidirectional replication of their chromosome(s) from a single locus known as the chromosome origin (oriC). At oriC the DNA double helix is opened to allow the loading of two replicative helicases around single-stranded DNA (ssDNA), one enzyme per strand. The initiation of bacterial DNA replication requires the formation of a nucleoprotein complex between oriC and the universally conserved master initiator DnaA. 
While DnaA protein sequence, structure, and activities are highly conserved, bacterial chromosome origins are diverse. The heterogeneity and apparent functional redundancy in bacterial origin architecture have obscured our understanding of oriC structure and function, leaving key fundamental questions unanswered. What constitutes the core structure of a bacterial chromosome origin, the DNA sequence information necessary to specify the mechanical opening of the DNA duplex by DnaA? Beyond the core origin, what are the functions of other essential oriC sequence elements?
A comprehensive sequence dissection of bacterial replication origins in their native genomic context is needed to develop physiologically relevant structure-function models of oriCs. By iteratively employing a combination of forward and reverse genetic approaches to sequence interrogate the oriC of B. subtilis, we have for the first time determined the complete structure and function of a bacterial replication origin in its chromosomal context in vivo. This analysis has identified the core sequences necessary and sufficient for DnaA-dependent DNA replication initiation, assigned specific roles to indirectly essential oriC sequence elements, as well as determined rules for DnaA-box location that dictate DnaA activity. Based on these findings, a unifying model for bacterial chromosome origin structure is presented.