Topoisomerase action on short DNA duplexes reveals requirements for gate and transfer DNA segments.
Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50-60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.
Belotserkovskii
Boris P
BP
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3402, USA.
Arimondo
Paola B
PB
Cozzarelli
Nicholas R
NR
D000255:Adenosine Triphosphate / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D027081:DNA Gyrase / Q000737:chemistry*; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D008954:Models, Biological; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase