Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II.
A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.
Lee
M P
MP
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Sander
M
M
Hsieh
T S
TS
D000818:Animals; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology / Q000201:enzymology*; D004625:Embryo, Nonmammalian / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004250:DNA Topoisomerases, Type II