Figure 1. Topo I Reaction Products Resolved on Non-EB and EB Gels.
Reactions (20 ul) were terminated with 1% SDS, digested with proteinase K and extracted with
CIA. The final volume after addition of Loading Dye was approximately 26 ul. Two agarose
gels (1%) were prepared. The top gel was cast and run in the absence of EB and the bottom
gel cast with 0.5 ug EB/ml and electrophoresed in buffer containing 0.5 ug EB/ml. Gels were
run at 50v for 45-50 min and either stained with EB (non-EB gel) or destained with water (EB
gel) per protocols given above. The data show the positions of nicked open circular (OC) DNA
which is pHOT1 DNA containing at least one single stranded nick (all plasmids have a small
amount of nicked OC DNA). Topoisomers are relaxed DNA forms that resolve after incubating
with Topo I in the absence of any drugs; these topoisomers are fully circular and contain no
single stranded interruptions. The formation of topoisomers is diagnostic for strong Topo I
relaxation activity and demonstrate that the enzyme is showing excellent activity. To see this
result, a non-EB gel should be run. The EB gel (Panel B) is ideal for detecting Topo I cleavage
products, such as nicked open circular DNA. The topo II active drug, VP16, did not influence the
topo I reaction, as expected. Note that supercoiled DNA substrate and relaxed DNA products
are rather poorly resolved in EB gels. In some cases, it is very difficult to demonstrate topo I
catalytic activity in this gel system as a result. For this reason, we recommend using a Non-EB
gel separation in activity assays.
Fig. 1. Typical Topo II reaction products with marker DNAs. A 1% EB containing agarose gel was run.
Lane 1: kDNA Catenated DNA marker. Lane 2, kDNA digested with Xho1. Lane 3, kDNA + 4 units topo II with 50 uM Etoposide.
Lane 4, same as lane 3 without etoposide. Red star marks the position of a topo II cleavage product (linear DNA).
This band is not seen in the absence of a topo II poison.