The Effect of Bleomycin on Sv40 Dna: Characteristics of Bleomycin Action Which Produces a Single Scission in a Superhelical Form of Sv40 Dna

Abstract

The effect of bleomycin on the molecular integrity of SV40 DNAwas determined under various conditions by taking advantage of the fact that a single scission produced in such a superhelical form of DNAcould be clearly manifested by analysis of samples with centrifugation in alkaline sucrose gradients. Conditions were found where the reaction velocity, the rate of disappearance of the characteristic form of DNA,depended on the concentration of bleomycin as well as on the reaction time. In contrast to general enzymatic reactions, bleomycin activity was greater at 0°C than at 37°C. The optimum pH was 9.1 while no activity was observed below pH 6 or above pH 13. Additional DNA,irrespective of its source, inhibited the reaction, while RNAhad no effect. Cu++ion at 1 mMalmost completely inhibited the reaction while 2-mercaptoethanol at 1 mMstimulated the reaction by about 20-fold. No sulfhydryl compound was included in our standard assay system. EDTAat 10 mM showed no effect.Bleomycin treatment produces a scission of DNAstrands both in vivo and in vitrol~~6\ In this action, bleomycin neither requires the native conformation^nor a particular base composition of DNA18). The fragmentation ofDNAby bleomycin, which can be enhanced by addition of reducing or oxidizing agents8'9*, is accompanied by release of free bases10) and aldehyde functions3). However, the detailed reaction sequence remains to be elucidated. To characterize bleomycin action, it seemed of primary importance to find reaction conditions where the rate of DNAfragmentation could conveniently be followed as a function of time and drug concentration. Use of SV40DNAas substrate is of major advantage since its native structure is double-stranded, covalently closed-circular and superhelical. It sediments with 53S at an alkaline pH, and if a single scission is produced on either strand of the native form, the products sediment with 18S (single-stranded, closed-circular DNA)and 16S (single-stranded, linear DNA) under the same conditions11*. Under restricted conditions, 16S and 18S DNAwere found to be the major products of bleomycin action because amounts of slower sedimenting DNAfragments or acid soluble material were negligible. Since bleomycin can act efficiently on DNAeven at 0°C, as will be described below, a critical aspect of our assay system was the use of an extremely alkaline pH to terminate the reaction. With this assay, reaction velocities as a function of bleomycin concentration were determined as was the effect of temperature, pHand various additives on the bleomycin activity. In the present paper, these results are presented and the probable mechanismof bleomycin action is discussed.

Binding of bleomycin to DNA: intercalation of the bithiazole rings

“…One test for an intercalator is its ability to relax supercoiled DNA without nicking the DNA strand (Waring, 1970). At low pH, bleomycin binds to DNA, but its nicking activity is greatly inhibited (Umezawa et al, 1973;Chien et al, 1977). When ColEl supercoils were sedimented in gradients at pH 5.5, presence of bleomycin caused a movement of the supercoil peak to coincidence with that of nicked circles (Figures 2 and 3A).…”

Properties and products of the degradation of DNA by bleomycin and iron(II)

“…The dependence of the reaction of DNA with Fe(II) and bleomycin on pH differs from the pH dependence observed in the absence of reducing agents (Umezawa et al, 1973) and in the presence of Mg(II)-ATP (Takeshita et al, 1977). In both of these studies substantial activity below pH 7 was not observed, in contrast to our results.…”

Light-induced nicking of deoxyribonucleic acid by cobalt(III) bleomycins

“…In several studies, divalent metal ions (including cobalt) that compete with iron for the same binding sites on BLM have been found to suppress the cleavage activity of BLM on isolated DNA (Suzuki et al, 1970;Umezawa et al, 1973;Takeshita et al, 1976; Lown & Sim, 1977;Sausville et al, 1978). It is nevertheless possible that metallobleomycins other than iron bleomycin may induce cleavage of DNA under conditions that are somewhat different from those used previously.…”