Abstract
The interaction of topoisomerase II with its DNA cleavage site is critical to the physiological functions of the enzyme. Despite this importance, the specific enzyme-DNA interactions that drive topoisomerase II-mediated DNA cleavage and religation are poorly understood. Therefore, to dissect interactions between the enzyme and its cleavage site, abasic DNA lesions were incorporated into a bilaterally symmetrical and identical cleavage site. Results indicate that topoisomerase II has unique interactions with each position of the 4-base overhang generated by enzyme-mediated DNA cleavage. Lesions located 2 bases 3' to the point of scission stimulated cleavage the most, whereas those 3 bases from the point of scission stimulated cleavage the least. Moreover, an additive and in some cases synergistic cleavage enhancement was observed in oligonucleotides that contained multiple DNA lesions, with levels reaching >60-fold higher than the wild-type substrate. Finally, topoisomerase II efficiently cleaved and religated a DNA substrate in which apyrimidinic sites were simultaneously incorporated at every position on one strand of the 4-base overhang. Therefore, unlike classical DNA ligases in which base pairing is the driving force behind closure of the DNA break, it appears that for topoisomerase II, the enzyme is responsible for the spatial orientation of the DNA termini for ligation.
Highlights
The interaction of topoisomerase II with its DNA cleavage site is critical to the physiological functions of the enzyme
Reactions contained 100 nM oligonucleotide in 19 l of cleavage buffer (10 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 100 mM KCl, and 2.5% glycerol) that contained 5 mM MgCl2 and were initiated by the addition of 1 l of human topoisomerase II␣. (Human topoisomerase II␣ was purified from Saccharomyces cerevisiae as described previously [19].) Reactions were incubated for 10 min at 37 °C and stopped with 2 l of 10% SDS followed by 1.5 l of 250 mM EDTA
To dissect the recognition of the DNA cleavage site by topoisomerase II and the role of base pairing in the cleavage/religation reaction of the enzyme, abasic lesions were incorporated into a 40-base pair oligonucleotide that corresponds to residues 1072–1111 of the MLL gene and includes a topoisomerase II cleavage site at nucleotide position 1087 [21, 22]
Summary
From the Departments of ‡Biochemistry and §Medicine (Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146. The interaction of topoisomerase II with its DNA cleavage site is critical to the physiological functions of the enzyme. Eukaryotic topoisomerase II is an enzyme that is required for a number of indispensable nuclear processes, including DNA replication, recombination, and chromosome segregation [1,2,3,4]. It is the primary target for some of the most effective and commonly employed anticancer chemotherapy regimens used for the treatment of human malignancies [5,6,7,8,9]. In order for topoisomerase II to fulfill its pivotal role in any of these processes, it must create double-stranded breaks in the genetic material
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