Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation

Abstract

Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought.