Characterization of a pathway of genomic instability induced by R-loops and its regulation by topoisomerases in E. coli.

The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance.

R-Loop Detection in Bacteria.

Early evidence for R-loop formation in vivo came from the study of Escherichia coli topA (topoisomerase I; topo I) null mutants. Assays with plasmids to detect RNase HI-sensitive hypernegative supercoiling or R-looped DNA were used in vitro and in vivo to demonstrate R-loop formation. In addition, these R-loop-dependent topological modifications of plasmid DNA were shown to correlate with severe growth and gene expression inhibition in topA null mutants that could be corrected by RNase HI overproduction.

Supercoiling, R-loops, Replication and the Functions of Bacterial Type 1A Topoisomerases.

Type 1A topoisomerases (topos) are the only topos that bind single-stranded DNA and the only ones found in all cells of the three domains of life. Two subfamilies, topo I and topo III, are present in bacteria. Topo I, found in all of them, relaxes negative supercoiling, while topo III acts as a decatenase in replication.

Topoisomerases I and III inhibit R-loop formation to prevent unregulated replication in the chromosomal Ter region of Escherichia coli.

Type 1A topoisomerases (topos) are the only ubiquitous topos. E. coli has two type 1A topos, topo I (topA) and topo III (topB).