UC Berkeley

All organisms depend on a variety of oligomeric ATPase assemblies to carry out essential cellular processes ranging from proteolysis and membrane trafficking to signaling events and nucleic acid transactions. The onset of DNA replication is one such process, relying on dedicated, ATP-dependent initiation factors to coordinate origin recognition and replisome assembly. Although cellular initiators and the initiators of certain classes of double-stranded DNA viruses use a variety of different strategies to process replication origins, they all possess a common adenine nucleotide-binding fold belonging to the AAA+ family of ATPases. To better understand the origin processing mechanism of AAA+ initiators, I performed a series of biochemical and structural studies with the bacterial initiator DnaA. In one study, I used structure-guided mutagenesis, biochemical, and genetic approaches to show that different oligomeric conformations of DnaA play distinct roles in controlling the progression of initiation. In a second study, I showed both crystallographically and in solution that the AAA+ domains of a spiral DnaA oligomer bind and extend single-stranded DNA segments in a RecA-like manner. These structural insights, combined with a novel DNA melting assay, indicate that DnaA uses its AAA+ domain to directly open replication origins by an active, ATP-dependent stretching mechanism. The studies presented in this dissertation provide a new model for origin opening in bacteria that has implications for the origin processing mechanism of all initiators.