Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate.
The cleavage and religation reactions of eukaryotic topoisomerase II were studied by use of a 5'-recessed DNA substrate containing a strong recognition sequence for the enzyme. Cleavage of the DNA substrate was suicidal, that is the enzyme was unable to religate the cleaved DNA due to a release of DNA 5' to the cleavage position. With this substrate cleavage products accumulated with time in the absence of protein-denaturing agents, and the cleavage reaction was not reversible with salt. The suicide cleavage complexes contained a kinetically competent topoisomerase II enzyme as determined by the enzyme's ability to perform intermolecular ligation of the cleaved DNA to a free 3'-hydroxyl end on another DNA strand. The efficiency of the religation reaction depended on the ability of the religation substrate to base pair to the DNA in the cleaved enzyme-DNA complex. Higher levels of religation were obtained with dinucleotides than with long DNA substrates. Mononucleotides also were efficiently religated, indicating an ability of the enzyme to mediate religation without making contacts to a long stretch of nucleotides 5' to the cleavage position.
Anderson
A H
AH
Department of Molecular Biology and Plant Physiology, University of Aarhus, Denmark.
Sørensen
B S
BS
Christiansen
K
K
Svejstrup
J Q
JQ
Lund
K
K
Westergaard
O
O
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D012965:Sodium Chloride / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013329:Structure-Activity Relationship; D013950:Thymus Gland / Q000201:enzymology; D013997:Time Factors
D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride; D004247:DNA; D004250:DNA Topoisomerases, Type II