2.3.5.1 Enzymology of DNA Supercoiling. DNA topoisomers are circular molecules, which have iden-
tical sequences and differ only in their linking number. A group of enzymes, discovered by Jim Wang, can
change that linking number.^33 They fall into two classes: Class I topoisomerases effect integral changes in
the linking number, Lkn, whereas Class II enzymes inter-convert topoisomers with a step rise of
Lk 2 n. TopoisomeraseI enzymes use a ‘nick-swivel-close’ mechanism to operate on supercoiled
DNA. They break a phosphate diester linkage, hold its ends, and reseal them after allowing exothermic
(i.e.passive) free rotation of the other strand. Such enzymes from eukaryotes can operate on either left- or
right-handed supercoils while prokaryotic enzymes only work on negative supercoils. The products of
topoisomerase I action on plasmid DNA can be observed by gel electrophoresis, and show a ladder of
bands, each corresponding to unit change in Wras the supercoils are unwound, half at a time (Figure 2.31).
By contrast, Class II topoisomerases use a ‘double-strand passage’ mechanism to effect unit change in
the number of supercoils, Wr 2, and such prokaryotic enzymes can drive the endothermic supercoil-
ing of DNA by coupling the reaction to hydrolysis of ATP. These topoisomerases cleave two phosphate
48 Chapter 2
Figure 2.31 Topoisomers of plasmid pAT153 after incubation with topoisomerase I to produce partial relaxation.
Electrophoresis in a 1% agarose gel: Track 1 shows native supercoiled pAT153 (S1), supercoiled dimer
(S2) and nicked circular DNA (N); Track 2 shows products of topoisomerase I where Lkup to 14 can
be seen clearly
(Adapted from D.M.J. Lilley,Symp. Soc. Gen. Microbiol., 1986, 39 , 105–117. © (1986), with permission
from the Society of General Microbiology)