Date of Degree
Seth A. Darst
Biochemistry | Biophysics | Structural Biology
DNA REPLICAITON, CRYO-EM, LAMBDA PHAGE, ESCHERICHIA COLI
Faithful transmission of genetic information is requisite for the propagation of all life. DNA replication in each of the three domains of life requires the separation of double stranded DNA (dsDNA) into single stranded DNA (ssDNA) which then serves as a template for genomic duplication of each original DNA strand. Initiation of replication events occurs by tightly regulated processes during which specialized proteins are loaded at a specific locus within the genome, termed the origin of replication, in preparation of bidirectional replication events. A replicative helicase must be loaded or assembled on both strands of DNA at the origin to both initiate and accomplish DNA replication. Subsequently, the replicative helicase travels ahead of the advancing replisome, acting as a wedge to separate DNA strands. In E. coli, DnaB is the bacterial replicative helicase and exists as a closed, homohexameric ring that must be loaded onto ssDNA, which is essentially an infinitely long polymer. Interestingly, both E. coli (host) and bacteriophage λ DNA replication requires loading of E. coli DnaB at their respective origins (E. coli: oriC, λ phage: ori λ). DnaB capture by either the bacterial helicase loader (DnaC) or phage helicase loader (λP) is crucial for determining whether the bacterial or phage genome is replicated, and is therefore of significant interest as a determinant factor for the initiation of organism-specific DNA replication events.
Our work seeks to understand the mechanisms by which the bacteriophage λ helicase loader (λP) captures the host E. coli DnaB helicase and redirects delivery of the helicase to the phage origin (ori λ) for the initiation of λ phage DNA replication. Prior to our findings, a lack of atomic level structural detail of DnaB helicase capture stymied the ability to compare helicase loading mechanisms in bacteria and phage. However, shortly following our initial work, high-resolution insights of DnaB captured by DnaC enabled a comparison of the loading mechanism employed by bacteria to our working understanding of that employed by λ phage. This enabled a comparison of helicase capture by a loader, delivery to ssDNA and helicase closing events between each organism. We find that λ phage helicase loader λP has evolved to employ a highly efficient and radically different mechanism for capturing the DnaB helicase in a nearly identical conformation that DnaC capture induces. Additionally, we find substantial differences in the configuration of DnaB upon transition from a loader captured helicase to a loaded helicase primed for replication events, which occur by apparently distinct mechanisms that are significantly influenced by biochemical properties of each respective loader. Together, our work enables a deeper understanding of how the bacterial replicative helicase is captured by either λP or DnaC, and the role of each loader in initiation of organism-specific replication events.
Chase, Jillian D., "Structural and Biochemical Investigations of the Initiation of DNA Replication in Bacteriophage Lambda and Escherichia coli" (2022). CUNY Academic Works.
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