esters to produce an enzyme-bridged gap in both strands. The other DNA duplex is passed through the gap
(using energy provided by hydrolysis of ATP), and the gap is resealed.^34 DNA gyrase from E. coliis a spe-
cial example of the Class II enzyme. It is an A 2 B 2 tetramer with the energy-free topoisomerase activity of
the A subunit being inhibited by quinolone antibiotics such as nalidixic acid. The energy-transducing
activity of the B subunit can be inhibited by novobiocin and other coumarin antibiotics. We should point
out that such topiosomerases also operate on linear DNA that is torsionally stressed by other processes,
most notably at the replication fork in eukaryotic DNA.
Supercoiling is important for a growing range of enzymes as illustrated by two examples. RNA poly-
merase in vitroappears to work ten times faster on supercoiled DNA 0.06, than on relaxed DNA, and
this phenomenon appears to be related to the enhanced binding of the polymerase to the promoter
sequence. Second, the tyrT promoter in E. coliis expressed in vitroat least 100 times stronger for super-
coiled than for relaxed DNA, and this behaviour seems linked to ‘pre-activation’ of the DNA promoter
region by negative supercoiling.^35
2.3.5.2 Catenated and Knotted DNA Circles. Although Class II topoisomerases usually only effect
passage of a duplex from the same molecule through the separated double strands, they can also manipu-
late a duplex from a second molecule. As a result, two different DNA circles can be inter-linked with the
formation of a catenane(Figure 2.32). Such catenanes have been identified by electron microscopy and can
be artificially generated in high yield from mammalian mitochondria. Knotted DNAcircles are another
unusual topoisomer species, which are also formed by intra-molecular double-strand passage from an
incompletely unwound duplex (Figure 2.32).
2.3.6 Triple-Stranded DNA
Triple helices were first observed for oligoribonucleotides in 1957. A decade later, the same phenomenon was
observed for poly(dCT) binding to poly(dGA)poly(dCT) and for poly(dG) binding to poly(dG)poly(dC).
Oligonucleotides can bind in the major groove of B-form DNA by forming Hoogsteen or reversed
DNA and RNA Structure 49
Figure 2.32 Action of topoisomerase II (red) on singly supercoiled DNA: (i) double-strand opening; (ii) double-strand
passage; (iii) resealing to give (a) relaxed circle, (b) knotted circle and (c) catenated DNA circles