Fig. 1

Figure 1. Outline of the In Vivo Link Kit

Native, active Topo I or II

DNA

Theoretical reaction sequence of topoisomerase on genomic DNA in Nuclei: Topo ligation (left) and cleavage (right) equilibrium

Sarkosyl or SDS Lysis

Denatured topo molecule

DNA Bound covalently to topo (=cleavable complex))

DNA without topo plus denatured free topo

Free Protein

DNA+ Topo/DNA complexes

Load CsCl Gradient

Fractionate from bottom of tube, locate DNA containing Fractions by UV absorbance

DNA Peak

Free Protein

Measure Topo Concentration in the DNA Peak using Western Slot Blotting

Western Slot blot: Cells treated with topo target drug and probed with antitopoisomerase antibody.

Wesrtern blot on the left demonstrates that topo protein was detected in the peak associated with genomic DNA. Cells were treated with a topo poison in this case. The blot measures topo/DNA covalent complexes formed in vivo..

Western Slot blot: Negative control, cells not exposed to drugs; probed with antitopoisomerase Antibody.

This Wesrtern blot gave no topo signal in the DNA peak. This is the negative control from cells NOT treated with a topo poison. With both type I and II enzymes, background levels of topo are detected in the DNA peak.

DNA PEAK

1 2 3 4

Figure 1A. Measuring Topo I and II covalent complexes in HeLa Cells.

HeLa cells, (2x10⁷cells/100 mm plate per treatment), were either treated (lane 1, camptothecin, lane 4, etoposide) or not (negative controls lanes, 2 and 4) for 30 min at 37^oC under phyisological conditions of growth. The cells were lysed by addition of sarkosyl and the viscous lysate overlayed onto a CsCl Step gradient. The four gradients were centrifuged overnight and fractionated from the bottom. DNA peaks were located using A ₂₆₀ determinations on each fraction. Approximately 50 ul from each fraction was vacuum loaded onto a slot blot (Schleicher & Scheull). The topo I blot (Lanes 1 and 2) were probed with anti-topo I antibody; the topo II blot (Lanes 3 and 4) was probed with anti-topo II antibody. The immune complexes on the membrane were then illuminated with [¹²⁵ I] -Protein A. Blots were exposed for 2 hrs. The DNA Peak fractions contain the appropriate topoisomerase (type I for camptothecin; type II for etoposide).

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