SLIDE 10 Example: ¡knomed ¡DNA ¡
4978
Knotting by E. coli Topoisomerase 1
FIG.
micrographs
DNA knots. Purified knot preparations
described in Fig. 2 were coated with recA protein, mounted, and photographed in the electron microscope. Molecules shown here are magnified approximately
X 80,000. Beneath each micrograph is a
tracing of the knot showing the over- and under-passing strand at each node. Below the tracing is an idealized redraw- ing of the knot and an identification number, consisting of the number of nodes, with a subscript indicating the knot's place in a standard tabulation (24, 25). A subscript c indicates a composite knot, composed of two distinct prime knots tied into a single ring.
ers among the 8 seven-noded knot forms, and 10 of these were found among the 14 seven-noded knots observed. Including more complex forms, a total of 32 different knots were observed. Of the knot forms with two enantiomers, both were observed in 8 of the 9 cases in which more than one example of the knot was found. Knots were formed that had all (+)- or all (-)-nodes, and many had a mixture of node
- sign. Knots 71, 72, 73, 74r
and 7 5 contain either all (+)- or all (-)-nodes but differ from one another in their arrangement. Several knots were observed that have both (+)- and (-)- nodes in the same knot, including 41, 61, 62r 63, 6,, 76, 77, and the composite 7-noded knot (7,) that is made up of 41 and a trefoil. We analyzed the trefoil knots statistically for bias toward either enantiomer; the 3 nodes of a trefoil are either all (+)
(+)- and 33 (-)-trefoils, and we
conclude that (+I- and (-)-trefoils are present in a 50:50 mixture (p > 0.2). Trefoils made by topoisomerase I have already been analyzed this way (13), but the 6x174 substrate used here is nicked only once and in a unique location. The important conclusion from this random knot distribution is that the enzyme neither selectively constrains the DNA into a unique conformation nor inverts duplex nodes of only one sign (see "Discussion"). Temperature Effect on Knotting-The DNA knots shown in Figs. 1-3 were generated by topoisomerase I in reactions incubated at 52 "C. High temperature was tried originally because of its success in revealing the activity of E. coli topoisomerase I11 (29), but we were concerned about the applicability of results
- btained for topoisomerase I at a
temperature so much higher than used previously. However, we find that under the conditions used for knotting, which includes 30% glycerol to stabilize the enzyme, the optimum for many topoisomerase I reactions is 52 "C or higher. The temperature profile for relaxation and knotting is shown in
- Fig. 5 for a constant amount of enzyme. For relaxation of
negative supercoils, the optimum is 52 "C, but even at 62 "C it is
37 "C. For knotting, the reaction increases up to 52 "C and remains at that level even at 62 "C. Thus, the temperature optimum for knotting, while high, is not very different from that of other reactions of topoisom- erase I. Requirements for Complex Knotting-We arbitrarily define
a complex knot as
- ne which contains at least 5
- nodes. There
are two requirements for the generation of complex knots, a high reaction temperature and a large molar excess of topoi- somerase I. At 52 "C, 300 fmol of topoisomerase I were re- Electron ¡micrographs ¡(x80,000) ¡of ¡E. ¡coli ¡DNA ¡knomed ¡by ¡the ¡ac-on ¡of ¡ ¡ topoisomerase ¡I. ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡Image ¡from ¡Dean ¡et ¡al ¡(1985) ¡J. ¡Biol. ¡Chem. ¡ ¡ ¡ “the ¡striking ¡result ¡is ¡that ¡the ¡enzyme ¡produces ¡every ¡knot ¡theore-cally ¡possible” ¡