Bacterial chromosome replication: does precatenation occur in vivo?

Each cell must replicate and segregate its DNA for the process of cell division. During the replication of the circular chromosome present in Escherichia coli the unwinding of the two parental strands leads to the formation of positive supercoils ahead of the replication fork. If the replication fork is free to rotate this might turn into precatenanes, intertwining of the two newly replicated chromosomes, behind the fork. If not resolved, supercoiling ahead of the fork can stall replication, while precatenation will turn into catenation at the end of replication, and will prevent chromosome segregation. Bacteria have mechanisms for the resolution of these undesired topologies which are performed by type II topoisomerases: Topoisomerase IV (Topo IV) and DNA gyrase. Topo IV is known to resolve precatenanes and catenanes, and gyrase can remove positive supercoiling. Both enzymes act by transporting one double stranded DNA helix through a gap made in a second double stranded segment of DNA. Interestingly, there is no direct evidence for precatenane formation on chromosomal DNA in living cells. Therefore, the aim of this work was to develop a method to detect precatenation in the bacterial chromosome. We tried to answer the following questions: Does precatenation occur on the bacterial chromosome in vivo? Is it Topo IV or DNA gyrase that decatenates in vivo? Are there mechanisms other than Topo IV or DNA gyrase responsible for precatenane and catenane unlinking in E. coli? The method consisted of using site-specific recombination between two recombination sites recognized by ΦC31 integrase. Once the integrase binds to the sites and recombines, the segment between the two sites is excised as a circle. After DNA replication, two circles will be produced, one from each sister chromosome. If the sisters were precatenated, the two circles might be catenated. Therefore, detection of catenanes after recombination occurred would indicate the presence of precatenation on the chromosome. Catenanes were detected as products of site-specific recombination on plasmid DNA in vivo when both type II topoisomerases were inhibited, suggesting that both Topo IV and DNA gyrase can decatenate DNA in vivo. Site-specific recombination was then used to detect precatenation on a plasmid replication intermediate model and on the chromosome. Interestingly, the products of recombination on the replication intermediate model and on the bacterial chromosome were the monomer of the 2.5 kb circle and its dimer. Formation of the dimer suggested that the two sister chromosomes co-localize after replication fork has passed and that might be due to precatenation. However, precatenation was not directly detected. Optimization of the method is required to obtain direct evidence of precatenation. In additional work, the level of supercoiling (Lk-Lk0) of a 398 bp circle produced by Xer recombination between closely spaced psi sites was determined to be -1. This together with previous results allowed the total linkage change of Xer recombination at psi to be determined as +4. Thus, the Xer reaction is driven by loss of four negative supercoils. This result is fully consistent with the Holliday junction model for strand exchange by the Xer recombinases and all other tyrosine recombinases.