Drosophila topoisomerase II-DNA interactions are affected by DNA structure.
The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.
Howard
M T
MT
Lineberger Cancer Research Center, Chapel Hill, NC 27514.
Lee
M P
MP
Hsieh
T S
TS
Griffith
J D
JD
D000818:Animals; D003421:Crithidia / Q000235:genetics; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism*; D018105:DNA, Kinetoplast; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004330:Drosophila / Q000201:enzymology*; D004587:Electrophoresis, Agar Gel; D008854:Microscopy, Electron; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics
D004270:DNA, Circular; D018105:DNA, Kinetoplast; D004279:DNA, Viral; D004250:DNA Topoisomerases, Type II