Molecular mechanisms of bacterial DNA replication
Cell Biology
Final Report Abstract
The faithful duplication of genetic material is essential for the proliferation of all life. Despite its fundamental importance, we still do not have a comprehensive picture of how DNA replication is initiated, especially in the most abundant lifeform on earth, bacteria. This is in particular due to the fact that we still do not know the function of all the parts of the bacterial origin of replication, (i.e. the location on the bacterial chromosome where DNA replication initiates). In a first step of bacterial DNA replication initiation, a master initiator protein called DnaA binds to a DNA sequence motif in the origin, the DnaA-box. In bacterial chromosomal origins, several DnaA-boxes are always found close to the site where the DNA is first opened, and there, DnaA will trigger the separation of the DNA strand and recruit the further machinery required for replication. However, many bacteria also have a cluster of essential DnaA-boxes belonging to the origin which is not located directly adjacent to the unwinding site. Origins with such a layout are called bipartite. The function of distant DnaA-box clusters and the spatial relationship between DnaA-box clusters of bipartite origins are still open questions. In my project, I used Bacillus subtilis, a model bacterium with a bipartite origin, to answer these questions by genetically altering the origin and analyzing the recruitment of factors involved in replication to its different regions. Surprisingly, I found that the distant DnaA-box cluster still supports DNA replication initiation when artificially transferred to a site 1.600 times further away than in the native locus, at the other end of the chromosome. I compared the binding of proteins essential for DNA replication initiation between mutant Bacillus subtilis strains in which I removed essential origin sequences. I found that the essential initiation protein DnaB, recruited by DnaA-box bound DnaA molecules, cannot be recruited to the unwinding site when the distant DnaA-box cluster is deleted. This suggests that the distant cluster is specifically needed to recruit essential initiation proteins such as DnaB. Fittingly, I later found that cells can evolve to survive the lack of the distant DnaA-box cluster by mutating the gene producing DnaB, altering the capability of DnaB to bind to other initiation proteins and DNA. Altogether, my results uncover surprising flexibility in the spatial layout of a bacterial origin and provide new insight into origin function and minimal sequence requirements for bacterial DNA replication initiation.
Publications
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HU Knew? Bacillus subtilis HBsu Is Required for DNA Replication Initiation. Journal of Bacteriology, 204(8).
Schramm, Frederic D. & Murray, Heath
