Jakob Frimodt-Møller:
Initiation of replication in Escherichia coli: A comprehensive study in regulation of initiation and the interlink to chromosomal organization

Date: 10-07-2015    Supervisor: Anders Løbner-Olesen

The circular chromosome of Escherichia coli is replicated by two replisomes assembled at the unique origin and moving in the opposite direction until they meet in the less well defined terminus. The key protein in initiation of replication, DnaA, facilitates the unwinding of double-stranded DNA to single-stranded DNA in oriC. Although DnaA is able to bind both ADP and ATP, DnaA is only active in initiation when bound to ATP. Although initiation of replication, and the regulation of this, is thoroughly investigated it is still not fully understood. The overall aim of the thesis was to investigate the regulation of initiation, the effect on the cell when regulation fails, and if regulation was interlinked to chromosomal organization.

This thesis uncovers that there exists a subtle balance between chromosome replication and reactive oxygen species (ROS) inflicted DNA damage. Thus, failure in regulation of initiation, which leads to hyperinitiation, results in double-strand breaks when replication forks encounters single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. Thus, the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised.

Furthermore, we found preserved distances from oriC to the non-coding regulatory regions controlling the activity of the initiator protein DnaA, datA, DARS1, and DARS2, in Escherichia coli. The structural organization of oriC, datA, DARS1, and DASR2 were also conserved, with most of the differences found in the non-functional spacer regions between key protein binding sites. Deletion of datA, DARS1, or DARS2 has an effect on regulation of initiation but not on the doubling time compared to wild-type. None of the regions or any combination of them was found to be essential for E. coli. Still, cells deficient in DARS1, DARS2, DARS1 and DARS2, or datA were found to be less fit than the wild-type in both LB medium and during mouse colonization. The observed chromosomal symmetry indicates that the position of datA, DARS1, and DARS2 are important for correct fine tuning of regulation of initiation in E. coli. Indeed, we show that the chromosomal position of regions influence the regulation of initiation. Relocation of DARS1 to oriC or datA to terC results in an increased origin concentration compared to the wild-type. DARS2 located in the terminus or on a low-copy number plasmid results in a lower origin concentration and asynchronous initiation. The optimal DARS1 and DARS2 location was investigated using a novel transposon mediated approach. Here we find that the optimal DARS2 position is in fact the wild-type position. The same is not true for DARS1. We also show that the cell needs a copy of both DARS1 and DARS2 for proper regulation of initiation; i.e. DARS1 is a poor replacement for DARS2 and vice versa. Last we suggest that transcription has a negative effect of the activity of the non-coding regions.