Co-ordination of DNA replication initiation and chromosome segregation during sporulation

PhD ThesisThe essence of viability and fitness of an organism is dictated by proper transmission of the genetic material from the parent to the progeny. Therefore in all organisms, processes of DNA replication and chromosome segregation are highly coordinated to match growth and cell division. Spore development in the model organism Bacillus subtilis provides a tractable system for studying a number of cellular processes including DNA replication and chromosome segregation. In the early stages of sporulation, DNA replication initiation is tightly regulated so that a single cell replicates only once and the diploidy is maintained. This is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that interacts with the N-terminal domain of DnaA and inhibits initiation of DNA replication in diploid Bacillus subtilis cells committed to sporulation. However the underlying mechanism by which SirA regulates the chromosome copy number in sporulating cells remains elusive. In the study presented here, I report that the in vivo examination of GFP-SirA localisation indicates that the protein accumulates at the replisome in sporulating cells. Also taking advantage of the newly released SirA crystal structure in complex with the N-terminal domain of DnaA (domain I), I show that the SirA localisation is dependent on the integrity of the interface identified in the co-crystal structure. This suggests that SirA accumulation at the replisome is likely a through a direct interaction with DnaA. In order to test the significance of inhibition of reinitiation events during spore development, I examined the chromosomal origin segregation in sporulating cells defective in regulating DnaA. I found that cells that lack regulators of DnaA; SirA, Soj or YabA exhibit 3 reduced fidelity in chromosomal origin trapping in the forespore compartment. I also provide evidence that the origin trapping defect observed in the cells lacking DnaA-regulators; SirA and YabA is a direct result of loss of regulation of DnaA. Finally, I show that Soj plays a DnaA-independent role in chromosomal origin trapping, for which its ability to localise at the septum seems to be required