Date of Award
Doctor of Philosophy (PhD)
DNA replication is a fundamental process required for all forms of life. The steps of prokaryotic DNA replication were originally elucidated through studies in the bacterial model organisms Escherichia coli and Bacillus subtilis, but the details of this process have not been investigated in most bacteria, including mycobacteria. Mycobacteria include the human pathogen Mycobacterium tuberculosis, which is a major global health threat due to high infection rates, emergence of multi-drug resistance, and a lengthy treatment regimen. DNA replication is a druggable process and the study of DNA replication in mycobacteria will expand our understanding of the basic biology of mycobacteria and therefore better inform the design of novel therapeutics.
While many DNA replication enzymes are conserved across all bacteria, the proteins involved in protein-protein interacting and regulatory steps are not homologous, but rather functionally analogous, between E. coli and B. subtilis. Many bacteria, including mycobacteria, lack identified homologs of DnaB helicase loading systems as well as regulators of the chromosomal replication initiator protein DnaA, despite these proteins playing essential roles during DNA replication in model organisms.
In this study we discover that Rv0004, which is conserved throughout Actinobacteria but absent from E. coli and B. subtilis, is essential for DNA replication in mycobacteria. Rv0004 binds DNA independent of sequence and directly interacts with DnaB. Rv0004 can promote DNA replication by facilitating the interaction of DnaB with DnaA. We also identify a DnaA N-terminal domain-like (DANL) domain in Rv0004 that is structurally predicted to be similar to the protein-interacting domain of DnaA. The DANL domain is located within Rv0004’s conserved domain of unknown function 721 and contains a tryptophan that is important for Rv0004 activity. Since mycobacteria are one of many bacterial genera that do not contain homologs of the DnaC-DnaI helicase loading systems found in model organisms, the study of Rv0004 reveals a new paradigm of prokaryotic DNA replication that is not limited to mycobacteria. The study of Rv0004 also emphasizes bacterial diversity in even the most conserved biological processes.
Chair and Committee
Christina L. Stallings
Tamara L. Doering, Jeffrey P. Henderson, Scott J. Hultgren, Petra A. Levin,
Mann, Katherine, "Discovery and Characterization of Rv0004 as a Novel and Essential Part of the Mycobacterial DNA Replication Machinery" (2017). Arts & Sciences Electronic Theses and Dissertations. 1229.
Available for download on Tuesday, June 27, 2119