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Biochemistry 1983, 22, 300-306
Role of Deoxyribonucleic Acid Topology in Altering the Site/Sequence Specificity of Cleavage of Deoxyribonucleic Acid by Bleomycin and Talisomycin t Christopher K. Mirabelli,*,* Cheng-Hsiung Huang,’ and Stanley T. Crooke’
ABSTRACT: The effects of changes in the topological conformation of deoxyribonucleic acid (DNA) on the site/sequence-specific breakage of DNA by the antitumor antibiotics bleomycin (BLM) A2 and talisomycin (TLM) A have been investigated. In this study, the site/sequence specificities of breakage by these drugs were compared by using isolated (1) linear restriction fragments, (2) whole, linear (form 111) pBR 322 DNA, or (3) covalently closed, superhelical (form I) pBR 322 DNA. The specificities of drug-produced breaks using these forms of DNA as substrates as determined by DNA sequencing analyses are summarized as follows: (1) The sequence specificity of BLM on linear DNA differs from that of TLM. (2) The specificities of BLM- and TLM-induced cleavage of linear DNA differed from those observed when form I DNA was used as substrate. (3) Although some of the
drug-induced sequence-specificbreaks which occurred in linear DNA were also observed in form I DNA, a number of sitespecific breaks occurred in form I DNA that were not observed in linear DNA incubated with drug. (4) These site-specific cleavage sites peculiar to form I DNA were observed with both BLM and TLM. (5) With form I DNA as substrate, as the number of breaks produced by BLM A, per molecule of DNA increased, the extent of cleavage at the sequence-specific sites increased relative to that at the cleavage sites peculiar to form I DNA. These results indicate that the specificity of cleavage of DNA by these drugs is influenced in part by the topology of the substrate and may have important implications with respect to the mechanism by which these drugs interact with cellular DNA.
%e bleomycins (BLM’s) * are a group of glycopeptide antibiotics which were first isolated by Umezawa et al. (1966) as copper complexes from culture media of Streptomyces verticillus. BLM was found to be a potent antibiotic against a variety of microorganisms (Ishizuka et al., 1967) and active against several neoplasms in man, both as a single agent and in combination chemotherapy (Crooke & Bradner, 1976). The antitumor activity of BLM, and of a group of structurally related antibiotics, the talisomycins, is thought to be related to their ability to induce single-strand and double-strand DNA breaks (Suzuki et al., 1969; Haidle, 1971; Muller et al., 1972; Takeshita et al., 1974; Strong & Crooke, 1978; Mirabelli et al., 1980). In vitro, BLM-induced DNA breakage has been suggested to involve the complex formation of BLM, Fe(II), and oxygen, with the subsequent production of free radicals which cause DNA strand breakage (Lown & Sim, 1977; Sausville et al., 1976). Recently, much attention has been focused on the sitelsequence-specific nature by which BLM fragments DNA. Using isolated DNA, it has been demonstrated that BLM cleaves DNA at specific sites (Lloyd et al., 1978b) and that the dinucleotide sequences G-C and G-T are preferentially cleaved by the drug (D’Andrea & Haseltine, 1978; Takeshita et al., 1978). Results from our laboratory suggest that a greater degree of specificity than that provided by only two nucleotides is involved in the site-specific recognition and cleavage of DNA by BLM and TLM (Mirabelli et al., 1982a,b). The structural and/or conformational characteristics in the locale of the bithiazole moiety of these drugs are an important determinant of their sitelsequence specificity. The TLM’s, which are
distinguished from the BLM’s by the presence of two amino acids and a 4-amino-4,6-dideoxy-~-talose sugar moiety located near the bithiazole, have a different site/sequence specificity for cleavage of DNA than that evidenced by BLM (Mirabelli et al., 1979, 1982a-c). The ability of BLM to affect DNA in deoxyribonucleoproteins also appears to be dependent on its functional state. Crooke et al. (1975) reported that nucleolar DNA was 2030-fold more sensitive than nucleoplasmic DNA to degradation by BLM. Furthermore, in isolated nuclei, BLM has been reported to preferentially degrade DNA sequences in open or “active” chromatin (Kuo, 1981). DNA in cells may exist in different conformational forms which are organized into various types of higher ordered genomic structures such as nucleosomes and superhelical or solenoidal arrays of polynucleosomes (Finch & Klug, 1976; Hewish & Burgoyne, 1973; Olins & Olins, 1974; Renz et al., 1979; Worcel, 1977). Thus, we have considered it important to study the effects of DNA conformation on the degradative activity and specificity of cleavage by the bleomycins as such effects may have biological consequences. We have recently observed that the ss and ds breakage activities of BLM A2were affected by the topological conformation of isolated PM2 phage DNA (Huang et al., 1982). In this paper, evidence is presented that indicates that the specificity of cleavage by both BLM and TLM is influenced, in part, by the topological conformation of the DNA substrate. The implications of these findings with respect to the mechanism by which these drugs interact with cellular DNA are also discussed.
From the Bristol Baylor Laboratory, Department of Pharmacology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030. Received May 17, 1982. This work was supported in part by a grant from Bristol Laboratories and a grant (CA-10893-P12) from the National Cancer Institute. *Present address: Smith Kline and French Laboratories, Philadelphia, PA 19101. S.T.C. is also affiliated with the Department of Pharmacology, Baylor College of Medicine.
0006-2960/83/0422-0300$0 1.5010
Materials and Methods Chemicals and Enzymes. Bleomycin A2 and talisomycin A were obtained from the Bristol Laboratories, Syracuse, NY.
’
Abbreviations: BLM, bleomycin; TLM, talisomycin; Na,EDTA, disodium ethylenediaminetetraacetic acid; Tris, tris(hydroxymethy1)aminomethane; DNA, deoxyribonucleic acid: ss, single strand; ds, double strand; bp, base pair(s).
0 1983 American Chemical Society
D N A TOPOLOGY A N D BLEOMYCIN CLEAVAGE SPECIFICITY
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or two closely spaced single-strand breaks which occurred in Restriction enzymes MspI and AvaI were purchased from New complementary strands within the “DNA breathing” distance. England Biolabs, Inc., Beverly, MA, DNA polymerase I (large Therefore, for location of the initial sites of damage, form I fragment) was from Boehringer Mannheim, Biochemica, West DNA was incubated with either TLM A or BLM A, under Germany, and [ C Y - ~ ~ P I ~(specific C T P activity 300 Cilmmol) conditions in which the majority of DNA molecules contained was from Amersham, Arlington Heights, IL. on average one or less breaks. Briefly, form I pBR 322 DNA pBR 322 DNA Preparation. Native plasmid pBR 322 DNA (preparations contained >95% form I ) was incubated with was grown in and isolated from Escherichia coli strain JA221 various concentrations of either drug (see Materials and according to the procedure described by Clewell & Helinski Methods), and the resulting percentages of form I 1 and form (1970). I11 DNAs were determined from agarose gels and densitoDNA Breakage Assay. BLM A, and TLM A were added metry as described previously (Mirabelli et al., 1980). From to 6 pg of form I pBR 322 DNA (only preparations containing these data, the percentages of molecules expected to have 0, greater than 95% form I DNA were used in these experiments) 1,2, , . . n nicks per molecule were determined by the Poisson in a buffer containing 10 mM Tris-HC1 (pH 7 . 9 , 20 mM distribution (C. K. Mirabelli et al., unpublished experiments). NaCl, and 40 mM dithiothreitol in a final volume of 120 pL. While the Poisson distribution does not accurately reflect this This solution was incubated for 30 min at 37 OC after which situation in which the breaks produced by BLM and TLM are 20 pL of the reaction mixture was added to 20 pL of a solution more than likely “nonrandom”, it does allow for a measure containing 20 mM Na2EDTA, 70% glycerol, and 0.05% (w/v) of the comparative breakage frequency by these two agents. bromphenol blue. Twenty microliters of this mixture was electrophoretically separated in a 1% agarose gel (Strong & Under the experimental procedures used, it was determined Crooke, 1979). The relative amounts of the various forms of that BLM A, and TLM A, at concentrations of 50 nM, plasmid DNA in each gel lane were determined as described produced on average 0.86 and 0.69 breaks per molecule of previously (Mirabelli et al., 1980). DNA, respectively. The DNA treated under these conditions In experiments in which the drugs were incubated with was then digested with the restriction enzyme AuaI, the 3’ isotopically labeled restriction fragments and whole linear pBR termini of the restricted fragments were labeled with 32Pand 322, the reaction conditions were equivalent to those described digested with MspI, and finally a 3’ end labeled 145-bp above. fragment of pBR 322 DNA was isolated. Using this proceSequence Analysis of BLMf TLM-Treated DNA. To the dure, the drug-produced single-strand nick sites which occurred remaining 100 pL of the drug-DNA reaction mixture deon the strand of DNA which was now labeled with 32Pwere scribed above was added EDTA to a concentration of 10 mM, localized by analysis on denaturing polyacrylamide-DNA and the DNA was then precipitated in 70% ethanol. The sequencing gels. Thus, the sites of damage that occurred when DNA was then digested with the restriction enzyme AuaI either form I DNA or linear DNA was incubated with drug which cleaves pBR 322 DNA at one site on the plasmid. The could be directly compared. DNA was isotopically labeled at the two 3’-terminal ends Figure 1 shows an autoradiograph of a sequencing gel produced by the AvaI digestion, using DNA polymerase I containing DNA which had been incubated with drug, in either (large fragment) (Setlow et al., 1972) and [ c Y - ~ ~ P I ~ C T P the . linear or the superhelical form, and the densitometricscans Experiments were performed verifying the results reported by of the lanes within the areas labeled A, B, and C are shown Niwa & Moses (1981) that the sites of damage in DNA in Figure 2a,b. In the scan of the linear DNA control, lane produced by BLM are not substrates for the incorporation of 1 (scan l), several minor peaks are evident, demonstrating the nucleotides by DNA polymerase I . The isotopically labeled existence of a small amount of cleavage within the untreated DNA was then digested with MspI, and the resulting 3’ end DNA fragment. The scan of the superhelical (form I ) DNA 32P-labeled145-bp restriction fragment was isolated and pucontrol (scan 9) shows that cleavages occurred at sites similar rified from 5% polyacrylamide gels (Maxam & Gilbert, 1980). to those in the linear DNA control but were relatively more The nucleotide sequences of restriction fragments and oliextensive. [These two control scans were measured at a 40% gonucleotides produced by BLM A2 and TLM A were dehigher densitometer gain setting than those shown in scans 2 termined by the method of Maxam & Gilbert (1980). Auand 3 (Figure 2a) and 7 and 8 (Figure 2b).] It should be noted toradiography was done by exposing Kodak XRP-5 film to that following the initial incubation of form I DNA in the the polyacrylamide gels for 8-48 h at -20 “C.The sequence absence of drug and subsequent isolation, approximately of the oligonucleotides was determined by comparison with 10-12% was determined to be converted to the nicked relaxed the chemically degraded restriction fragments, the nucleotide form I 1 configuration. Therefore, the bands present in lane sequences of which have been reported by Sutcliffe (1978). 9 most likely represent the nicks produced during this incuAutoradiographs were scanned with a scanning densitometer bation and/or during the subsequent isolation procedure. The (RFT Model 2955 scanning densitometer, Transidyne General experimental difference between the control DNA lanes in C o p ) and the relative positions and amounts of the 32P-labeled Figure 1 is that the DNA in lane 9 was incubated in drug oligonucleotides determined. reaction conditions (minus drug) as form I DNA, whereas the DNA in lane 1 was incubated under these conditions as the Results isolated 145-bp restriction fragment. The production of single-strand breaks and double-strand The amount of cleavage observed in DNA fragments which breaks in covalently closed superhelical form I DNA by were obtained from samples incubated in the absence of drug treatment with bleomycin can be accurately measured by gel varied from experiment to experiment. However, upon relaelectrophoresis of the form I 1 and form I11 DNAs, respectively tively extended exposure times, cleavage was always observed (Lloyd et al., 1978a,b; Johnson & Grossman, 1977; Strong in the autoradiographs. & Crooke, 1978; Mirabelli et al., 1980; Huang et al., 1981). Scan 3 in Figure 2a shows that cleavage of form I DNA Only the first single-strand break in each DNA molecule is treated with BLM A2 under conditions which produced an detected since a single break is sufficient to relax the superaverage of 0.86 break per DNA molecule occurred at a number helical DNA to form I 1 DNA. The production of doubleof different sites, many of which were also nicked to a relatively strand breaks can be a result of a direct double-strand break
302 B I O C H E M I S T R Y
MIRABELLI, H U A N G , A N D C R O O K E
a
l-~&--i-c-l
c i
mom 1: Sequencing gel analysis of drug-pmduad oligonucleotides obtained when either form I or a 145-bp restriction fragment of pBR 322 DNA is used as the substrate (1) (see text for explanation of expnimental procedurs). This figure is an autoradiograph of an 8% sequencing gel (Materials and Methods). Each lane was loaded with equivalent total amounts of cpm of sample. Lanes 1. 2, and 4-1 correspond to reactions with isolated 31P-labeled restriction fragment. Lanes 3,8, and 9 contain DNA which was incubated as form I DNA in the presence or absence of drug, after which the DNA was 32P labeled and the 145-bprestriction fragment isolated. (Lane 1) Control (no drug); (lane 2) 200 p M BLM A,; (lane 3) 50 nM BLM A,; (lane 4) T + C (basespecific chemical reactions); (lane 5 ) C (base-specific chemical reactions); (lane 6 ) A > C (base-specific chemical reactions); (lane 7) 200 pM TLM A; (lane 8) 50 nM TLM A, (lane 9 ) (no drug) control. All BLM, TLM, and control reactions were done under standard drug reaction conditions (Materials and Methods). The sequence of the restriction fragment is read from lanes 4-6. starting at the bottom of the gel in the 3’ to Sdirection. The nucleotide sequence and the distance (in base pairs) from the 32Plabeled 3’ end of the fragment are indicated at the left. Areas labeled A, B, and C (right side) are depicted as densitometric scans in Figure 2a,b.
1111111111111I C1 L1 A1 C1 C1 C1 T1 T1 G1 G1 cI *I Gl * l
oToI*Tocrlc**Ac
lesser extent within the control form I DNA (scan 9). For example, withiin the area labeled B, there are two major peaks and three minor peaks in the scan corresponding to the BLM A, treated form I DNA. The scan of the form I DNA, incubated in the absence of drug, reveals peaks at similar pasitions, although they did not have the same relative peak areas as those in scan 3 (scans 1 and 9 were obtained at 40% higher gain than scans 2 and 3). Similar comparisons of scans 9 and 3 can be seen in areas labeled A and C. The peaks in scan 2 in Figure 2a correspond to the cleavages which occurred in the linear DNA treated with BLM A,. The cleavages occurred predominantly in areas A and C and correspond to the sequence-specific sites described previously (Mirabelli et al., 1982a.b). These cleavages occurred at positions different from those produced in form I DNA treated with BLM A, or generated in the absence of drug. Results similar to those discussed above for BLM A, were
I 70
rn
(a) Densitometric scans of lanes in Figure 1. Numbers to scans correspond to lane numbers. Areas labeled A, B, and C and the nucleotide sequence correspond to respective areas in Figure 1. (b) Densitometric scans of lanes in Figure 1. Numbers to scans correspond to lane numbers. Areas labeled A. B. and C corresmnd to respective labeled areas in Figure I ~ O U R E2
observed with the superhelical and linear DNA species treated with TLM A. Superhelical DNA treated with TLM A was cleaved at sites equivalent to those nicked in form I DNA incubated in the absence of drug (Figure 2b, scans 8 and 9, respectively). These shared cleavage sites were more extensively cleaved in the superhelical DNA treated with TLM relative to the control. The linear DNA was cleaved at se quencaspecific sites which did not correspond with those sites cleaved in the superhelical DNA (scans 7 and 8, respectively). As reported previously, TLM A cleaved the linear restriction
D N A TOPOLOGY A N D B L E O M Y C I N C L E A V A G E S P E C I F I C I T Y
fragment at specific sites different from thaw. cleaved by BLM A2 (Mirabelli et al., 1979, 1982~).Sequence analyses demonstrate that TLM cleaved at G-T and G-C sequences. However, many of these particular sequences were cleaved to significantly different extents by TLM relative to BLM (Mirabelli et al., 1982a.b). G-A sequences were cleaved preferentially by TLM while they were relatively resistant to cleavage by BLM (Figure 1). Experiments were then conducted to determine if there was a change in the cleavage site preference of either drug as the amount of breakage on the plasmid increased. Following the fmt single or doublestrand break in a f a n I DNA molecule, the subsequent breaks in the molecule would result from the interaction of the drugs with the DNA in either a form I1 or a form I l l conformation. Therefore, form 1DNA was incubated with various concentrations of drug, and aliquots of the resulting DNA were analyzed on agarose gels to determine the amount of damage p r c d u d . The DNA was then digested with restriction enzymes and isotopically labeled, and the 144-bp restriction fragment was isolated as described under Materials and Methods. These fragments were then analyzed for nicks on sequencing gels as shown in Figure 3. The scans of lanes 1-5 and 8-10 are shown in panels a and b, respectively, of Figure 4. The areas of the scans, labeled A, B, and C, mespond to the equivalent areas in the DNA fragment shown in Figure 2a.b. A scan of lane 1 in Figure 3 shows that the form I DNA incubated in the absence of drug contained Ncks, the most predominant of which were located in areas B and C. This scan was measured at a 60% higher densitometric gain setting than those shown in scans 2-5 in Figure 4a and scans 9-1 1 in Figure 4b. Scans 3.4, and 5 show the cleavage pattern produced when form I DNA was incubated with BLM A2 under conditions which produced 1.6.5.0, and
