Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate.
Following its cleavage of double-stranded DNA, topoisomerase II is covalently bound to the 5'-termini of both nucleic acid strands. However, in order to isolate this enzyme-cleaved DNA complex in the presence of magnesium (the enzyme's physiological divalent cation), reactions must be terminated by the addition of a strong protein denaturant such as sodium dodecyl sulfate (SDS). Because of the requirement for a protein denaturant, it is unclear whether DNA cleavage in this in vitro system takes place prior to or is induced by the addition of SDS. To distinguish between these two possibilities, experiments were carried out to determine whether topoisomerase II bound DNA contains 3'-OH termini prior to denaturation. This was accomplished by using circular single-stranded phi X174 DNA as a model substrate for the enzyme. As found previously for topoisomerase II mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5'-termini of cleaved phi X174 molecules. Moreover, optimal reaction pH as well as optimal salt and magnesium concentrations was similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not salt reversible, and did not require the presence of SDS. Furthermore, cleavage products generated in the absence of protein denaturant could be labeled at their 3'-OH DNA termini by incubation with terminal deoxynucleotidyltransferase and [alpha-32P]ddATP. Finally, cleaved phi X174 molecules could be joined to a radioactively labeled double-stranded oligonucleotide by a topoisomerase II mediated intermolecular ligation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Gale
K C
KC
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Osheroff
N
N
D000818:Animals; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology; D004625:Embryo, Nonmammalian / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D011865:Radioisotope Dilution Technique; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D004250:DNA Topoisomerases, Type II