DNA double-strand break repair and the termination of replication in Escherichia coli

Faithful DNA replication is essential for the maintenance of genetic information. This complex process consists of 3 steps: initiation, elongation and termination. Although the first two steps are quite well understood in both eukaryotes and prokaryotes, many aspects of the termination of replication remain unclear. Escherichia coli is an ideal organism to study termination of DNA replication. In E. coli, DNA replication starts by bidirectional firing of two replication forks from a unique origin and terminates when those forks collide in the terminus region of the circular chromosome. The terminus region is flanked by specific ter sequences, which ensure that termination of replication occurs within specific boundaries. Due to the circularity of the E. coli chromosome, once the replication is finished the dimers can be formed. To resolve the dimers, the dif sequences are aligned together and two chromosomes are then separated into two daughter cells. Previous members of Prof. Leach laboratory have observed a stimulation of both double-strand break repair (DSBR) and DNA over-replication in the terminus region when DSBR was induced in the lacZ locus, half way between the origin and the terminus. In this work, I propose that these two phenomena, elevated levels of DSBR and DNA over-replication, are linked to each other. I confirm that the DSBs arise from the dif site and that the dif site is the source of DNA over-replication in the terminus. My results suggest that an attempted DSBR at dif leads to over-replication between terA and terB. Here, using next generation sequencing methods, I show that TopoIV and TopoIII topoisomerases introduce breaks in chromosome dimers that were not resolved by the XerCD/dif system, leading to DSBR and DNA over-replication.