Structural and Biochemical Studies of Replicative Helicase Loading in Bacteria

The initiation of DNA replication represents a defining commitment to the proliferation of all cellular organisms. Dedicated ATP-dependent initiation factors specifically mark replication start sites or “replication origins” and, together with ATP-dependent helicase loaders, help coordinate productive assembly of the replisome. In many bacterial species, helicase loaders belonging to the DnaC/I family of proteins assist the bacterial initiator factor DnaA with loading and activation of DnaB-type replicative helicases. Despite advances in understanding some of the essential proteins and common principles underlying initiation strategies, major questions still persist in defining how initiation programs are executed at the molecular level./nThe present dissertation presents a combination of structural and biochemical studies of the DnaC helicase loader and DnaA initiator from the Gram-negative bacterium Escherichia coli (E. coli) and the DnaI helicase loader from the Gram-positive bacterium Staphylococcus aureus (S. aureus). For the E. coli work, I developed several fluorescence-based helicase assays to help define the particular contributions of both DnaA and DnaC in promoting loading of DnaB at a replication origin. Investigations of DnaA mutants in this helicase assay revealed the importance of the initiator’s N-terminal helicase binding domain in recruiting DnaB to a nascent bubble or fork to facilitate helicase loading. Parallel studies of the E. coli DnaC demonstrated: (i) that the loader’s N-terminal DnaB binding domain is sufficient for activation of E. coli DnaB’s duplex DNA unwinding activity and (ii) that the AAA+-family ATP binding and hydrolysis domain of DnaC likely serves as a regulatory element that helps enhance the efficiency of helicase activation./nTo better characterize the activity of a Gram-positive S. aureus DnaI helicase loader, I structurally and biochemically characterized the protein’s ATPase domain and activity, and also investigated how a particular viral peptide inhibitor from phage 77, termed “ORF104”, interferes with host DNA replication by blocking SaDnaI activity. Comparative structural analyses, combined with biochemical studies of SaDnaI revealed not only how the viral inhibitor 77ORF104 blocks loader function but also revealed insights into how bacterial helicase loaders may in general auto-regulate their function. A complimentary viral-host interaction study of another staph-specific viral peptide that also inhibits host DNA replication (“ORF078” from phage 71) has revealed another promising anti-bacterial strategy: preliminary binding studies indicate that this gene product binds primase through its helicase-binding domain./nOverall, the studies presented in this dissertation provide multiple new insights into fundamental mechanisms underlying the initiation of DNA replication. One is an enhanced understanding of the role that the E. coli DnaA initiator and DnaC loader serve in recruiting the DnaB helicase to a replication origin. The other describes a novel viral mechanism for inhibition of the S. aureus DnaI helicase loader that helps establish how a virus may exploit an existing auto-regulatory element of the bacterial helicase loader as part of a strategy to inhibit host DNA replication. These findings collectively not only answer long-standing questions in the field, but also open up new avenues for future inquiry.

Advisor: Berger, James M