Replication dynamics in double origin E. coli strains. oriZ replication profiles

Each time a cell divides millions of DNA base pairs have to be unwound to allow accurate duplication of the genetic material as well as transcription of the necessary genes. DNA replication and transcription have to proceed simultaneously on the same template, and conflicts between DNA and RNA polymerases are unavoidable. Head-on collisions are thought to be particularly problematic. Thus, it came as a surprise that a recent study reported unperturbed replication fork and cell cycle progression in Escherichia coli cells with an ectopic replication origin, as a number of highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found that the doubling time to be at least twice that of normal cells, indicating serious problems. Replication profiles revealed that both highly transcribed regions as well as the termination area show significant distortions, which can be alleviated by point mutations that either inactivate the termination area or destabilise RNA polymerase complexes, allowing easier removal by traversing replication forks. Analysis of the replication profile of the previously used E. coli construct revealed a chromosomal rearrangement which alleviates the above problems in an intriguingly simple way. Our data demonstrate that head-on collisions of DNA replication and transcription can be highly problematic, supporting the idea that avoiding such conflicts has resulted in the high level of co-directionality of highly transcribed genes and DNA replication observed in many bacterial chromosomes.