Comparison of the Sequences at Specific Sites on DMA Cleaved by the Antitumor Antibiotics Talisomycin and Bleomycin1

We have investigated the site-specific cleavage of DNA by the antitumor antibiotics talisomycin and bleomycin by using 5'or 3'-terminal 32P-labeled restriction fragments of pBR 322 DNA. Both drugs cleaved DNA preferentially at G-C and G-T sequences. However, the relative amounts of cleavage at par ticular cleavage sites differed between talisomysin and bleo mycin at concentrations of the drugs which produced similar extents of total cleavage. In addition, talisomycin produced specific cleavages at G-A sequences which were relatively resistant to cleavage by bleomycin. Within a preferred se quence group (i.e., G-C sequences), some sites were cleaved to a greater extent relative to others by both talisomycin and bleomycin, suggesting that a greater degree of specificity than that provided by only two nucleotides is involved in the sitespecific recognition and cleavage of DNA by these drugs. INTRODUCTION The BLMs3 are a group of glycopeptide antibiotics, isolated from Streptomyces verticillus (29) that have been shown to be effective against a variety of neoplasms (3). The primary target for BLM cytotoxicity (3) appears to be interaction with cellular DNA. The effects of BLM on isolated DNA have been shown to include liberation of free bases (5, 20), site-specific and non specific singleand double-strand breakage (4-6, 10, 11, 14, 19, 22, 28), noncovalent intermolecular cross-links (12), and a reduction of DNA melting temperature (21). BLM produced breakage of DNA in cells grown in tissue culture, and the extent of degradation was correlated with cell cycle-specific cytotox icity of the drug (1 ). Recently, it has been reported that BLM can preferentially degrade the DNA sequences in the open chromatin within isolated nuclei (9). TLM are a group of antitumor antibiotics related structurally to the BLMs. The TLMs contain 2 new amino acids and a unique sugar, 4-amino-4, 6-dideoxyl-L-talose that have not been found previously in the BLM complex (8). TLM A has exhibited significantly greater antibiotic activity against a vari ety of bacteria and fungi than did BLM A2 (7). The 2 major components of the antibiotic, TLM A and TLM B, have shown antitumor activity in experimental animal tumor systems (7). Like BLM, TLM has been shown to cause DNA breakage in cells grown in tissue culture and singleand double-strand breaks in isolated DNA (13,17, 25). However, their relative cell 1This work was supported in part by a grant from Bristol Laboratories and Grant CA-10893-P12 from the National Cancer Institute. 2 To whom requests for reprints should be addressed. (Present address; Smith Kline & French Laboratories, (F124), 1500 Spring Garden St., P. O. Box 7929, Philadelphia, PA 19101.) 3 The abbreviations used are: BLM, bleomycin; TLM. talisomycin. Received August 18, 1981; accepted January 5, 1982. cycle specificities (16) and singleand double-strand breakage activities appear to be different (17). We have shown recently that the site specificities of doublestrand fragmentation of DNA by the 2 antitumor antibiotics appear to differ (19). The purpose of the experiments presented here was to determine the nucleotide sequences at the TLMspecific cleavage sites and to compare these with the sites cleaved by BLM. MATERIALS AND METHODS Materials. Copper-free BLM A2 and TLM A were obtained from Bristol Laboratories, Syracuse, N.Y.; restriction enzymes Ava\ and Msp\ were purchased from New England Biolabs, Beverly, Mass.; bacterial alkaline phosphatase was obtained from Bethesda Research Laboratories, Rockville, Md.; polynucleotide kinase was obtained from P-L Biochemicals; DNA polymerase I (large fragment) was obtained from Boehringer Mannheim, Biochemica, W. Germany; and [y-32P}ATP (specific activity, ~5000 ci/mmol) or dCTP (specific activity, 3000 ci/mmol), were obtained from Amersham/Searle Corp., Arlington Heights, III. Covalently closed superhelical plasmid pBR 322 DNA was isolated from Escherichia coli strain Ja 221 according to the procedure of Clewell et al. (2). Preparation of DNA Restriction Fragments. With the use of stand ard reaction conditions, the isolated pBR 322 DNA was digested with Ava\ restriction enzyme. The 2 terminal ends of the resulting whole linear pBR 322 DNA were labeled by either of 2 methods: (a) the restricted DNA was incubated with bacterial alkaline phosphatase, and the two 5' termini were then rephosphorylated with [y-32P]ATP and polynucleotide kinase; (b) restricted DNA was isotopically labeled at the two 3'-terminal ends with DNA polymerase (large fragment) (24) and [<*-32P]dCTP. After either labeling procedure, the DNA was digested with Msp\. The resulting 145and 61-base-pair fragments (27) each labeled at one 5'-terminal end (Labeling Procedure 1) or at one 3'terminal end (Labeling Procedure 2) with 32Pwere isolated and purified from 5% polyacrylamide gels (15). Fragmentation of DNA by BLM and TLM. The reaction mixture contained 10 rriM Tris-HCI (pH 7.5), 10 rnw NaCI, 40 mw dithiothreitol, 5'-terminal 32P-labeled restriction fragments (10 ng, 50,000 cpm), 7 fig calf thymus DNA, and either BLM A2 or TLM A at concentrations ranging from 1.0 to 500 /IM in a total volume of 10 /il. After incubation for 30 min at 37°, 10 rtiM EDTA was added to stop the reaction, and the solution was immediately frozen and lyophilized. Each sample was dissolved in 10 /¿I of a solution of 80% deionized formamide:0.025% bromphenol blue:xylene cyanole. The solution was heated at 90° for 30 sec, and 3-/J aliquots were transferred to 8 or 20% polyacrylamide gels for sequence analysis (15). Sequence Analysis. The nucleotide sequences of restriction frag ments and oligonucleotides produced by BLM A2 and TLM A were determined by the method of Maxam and Gilbert (15). Autoradiography was done by exposing Kodak XRP-5 film to the polyacrylamide gels for 8 to 48 hr at -20°. The sequence of the oligonucleotides was deter mined by comparison of the chemically degraded restriction fragments,