POSITION-SPECIFIC EFFECTS OF BASE MISMATCH ON MAMMALIAN TOPOISOMERASE-II DNA-CLEAVING ACTIVITY

To further define the nucleic acid determinants of DNA site recognitio n by mammalian topoisomerase II, base mismatch effects on the enzyme D NA cleavage activity were determined in a 36-bp synthetic oligonucleot ide corresponding to SV40 DNA. DNA cleavage sites induced by topoisome rase II without or with the antitumor drugs teniposide, idarubicin, or amsacrine were mapped using sequencing gels. Selected mismatches were studied, and always one of the two strands had the wild-type sequence . The effects of base mismatches were independent from the studied dru gs. Mismatches introduced at the -4, -3, -2, or -1 positions, relative to the enzyme cleavage site, often abolished, or much reduced, DNA cl eavage, whereas those at +1 and +2 positions often increased DNA break age or were without influence. Mismatches at more distant positions, e .g., -7, -8, etc., had no effect. Those at positions -5 and -6 sometim es increased cleavage levels. These effects were always observed at si tes already cleaved in the wild-type oligomer; new sites of cleavage w ere not induced by the studied mismatches. These results were obtained both for the native murine topoisomerase II and for the two recombina nt human isozymes. No difference between topoisomerases II alpha(p170) and beta(p180) was seen in their response to mismatches. The results demonstrate that topoisomerase II recognition of the DNA site of cleav age requires fully paired nucleotides at the 3' terminus. Nevertheless , similarly to other DNA strand transferase enzymes, both topoisomeras e II isoforms may have a sequence-specific nicking activity at the 5' side of unpaired bases.