Detection of oriC-Independent Replication in Escherichia coli Cells.

In bacteria, replication of the chromosome is normally initiated following the binding of DnaA proteins to the oriC region. However, under certain circumstances, replication can also be initiated independent of the oriC/DnaA system. This is the case, for example, in Escherichia coli cells lacking RNase HI (rnha mutants) or type 1A topoisomerase activity (topA topB mutants). Here, we present a protocol in which replication from the oriC/DnaA system is first inhibited by the addition of the protein synthesis inhibitor, spectinomycin, to exponentially growing bacterial cell cultures. The thymidine analog, 5-ethynyl-2'-deoxyurdine (EdU) is then added to the cells, and after 1 h the cells are fixed and the Alexa Fluor 488 dye is conjugated to EdU by the click-iT reaction. The oriC-independent replication is detected in fixed cells by flow cytometry and can be visualized by fluorescence microscopy.