Role of DNA topology in altering the site/sequence specificity of cleavage of DNA by bleomycin and talisomycin

3 00 Biochemistry 1983, 22, 300-306

Role of Deoxyribonucleic Acid Topology in Altering the Site/Sequence Specificity of Cleavage of Deoxyribonucleic Acid by Bleomycin and Talisom ycin 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

%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 di- nucleotide 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

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 Pharma- cology, Baylor College of Medicine.

0006-2960/83/0422-0300$0 1.5010

drug-induced sequence-specific breaks which occurred in linear DNA were also observed in form I DNA, a number of site- specific 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.

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 20- 30-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 A2 were 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.

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

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Restriction enzymes MspI and AvaI were purchased from New England Biolabs, Inc., Beverly, MA, DNA polymerase I (large fragment) was from Boehringer Mannheim, Biochemica, West Germany, and [ C Y - ~ ~ P I ~ C T P (specific activity 300 Cilmmol) was from Amersham, Arlington Heights, IL.

pBR 322 DNA Preparation. Native plasmid pBR 322 DNA was grown in and isolated from Escherichia coli strain JA221 according to the procedure described by Clewell & Helinski (1970).

DNA Breakage Assay. BLM A, and TLM A were added to 6 pg of form I pBR 322 DNA (only preparations containing greater than 95% form I DNA were used in these experiments) in a buffer containing 10 mM Tris-HC1 (pH 7 .9 , 20 mM NaCl, and 40 mM dithiothreitol in a final volume of 120 pL. This solution was incubated for 30 min at 37 OC after which 20 pL of the reaction mixture was added to 20 pL of a solution containing 20 mM Na2EDTA, 70% glycerol, and 0.05% (w/v) bromphenol blue. Twenty microliters of this mixture was electrophoretically separated in a 1% agarose gel (Strong & Crooke, 1979). The relative amounts of the various forms of plasmid DNA in each gel lane were determined as described previously (Mirabelli et al., 1980).

In experiments in which the drugs were incubated with isotopically labeled restriction fragments and whole linear pBR 322, the reaction conditions were equivalent to those described above.

Sequence Analysis of BLMf TLM-Treated DNA. To the remaining 100 pL of the drug-DNA reaction mixture described above was added EDTA to a concentration of 10 mM, and the DNA was then precipitated in 70% ethanol. The DNA was then digested with the restriction enzyme AuaI which cleaves pBR 322 DNA at one site on the plasmid. The DNA was isotopically labeled at the two 3’-terminal ends produced by the AvaI digestion, using DNA polymerase I (large fragment) (Setlow et al., 1972) and [ c Y - ~ ~ P I ~ C T P . Experiments were performed verifying the results reported by Niwa & Moses (1981) that the sites of damage in DNA produced by BLM are not substrates for the incorporation of nucleotides by DNA polymerase I . The isotopically labeled DNA was then digested with MspI, and the resulting 3’ end 32P-labeled 145-bp restriction fragment was isolated and pu- rified from 5% polyacrylamide gels (Maxam & Gilbert, 1980).

The nucleotide sequences of restriction fragments and oligonucleotides produced by BLM A2 and TLM A were determined by the method of Maxam & Gilbert (1980). Au- toradiography was done by exposing Kodak XRP-5 film to the polyacrylamide gels for 8-48 h at -20 “C. The sequence of the oligonucleotides was determined by comparison with the chemically degraded restriction fragments, the nucleotide sequences of which have been reported by Sutcliffe (1978). Autoradiographs were scanned with a scanning densitometer (RFT Model 2955 scanning densitometer, Transidyne General C o p ) and the relative positions and amounts of the 32P-labeled oligonucleotides determined.

Results The production of single-strand breaks and double-strand

breaks in covalently closed superhelical form I DNA by treatment with bleomycin can be accurately measured by gel electrophoresis of the form I 1 and form I11 DNAs, respectively (Lloyd et al., 1978a,b; Johnson & Grossman, 1977; Strong & Crooke, 1978; Mirabelli et al., 1980; Huang et al., 1981). Only the first single-strand break in each DNA molecule is detected since a single break is sufficient to relax the superhelical DNA to form I 1 DNA. The production of double- strand breaks can be a result of a direct double-strand break

or two closely spaced single-strand breaks which occurred in complementary strands within the “DNA breathing” distance. Therefore, for location of the initial sites of damage, form I DNA was incubated with either TLM A or BLM A, under conditions in which the majority of DNA molecules contained on average one or less breaks. Briefly, form I pBR 322 DNA (preparations contained >95% form I ) was incubated with various concentrations of either drug (see Materials and Methods), and the resulting percentages of form I 1 and form I 1 1 DNAs were determined from agarose gels and densito- metry as described previously (Mirabelli et al., 1980). From these data, the percentages of molecules expected to have 0, 1,2, , . . n nicks per molecule were determined by the Poisson distribution (C. K. Mirabelli et al., unpublished experiments). While the Poisson distribution does not accurately reflect this situation in which the breaks produced by BLM and TLM are more than likely “nonrandom”, it does allow for a measure of the comparative breakage frequency by these two agents.

Under the experimental procedures used, it was determined that BLM A, and TLM A, at concentrations of 50 nM, produced on average 0.86 and 0.69 breaks per molecule of DNA, respectively. The DNA treated under these conditions was then digested with the restriction enzyme AuaI, the 3’ termini of the restricted fragments were labeled with 32P and digested with MspI, and finally a 3’ end labeled 145-bp fragment of pBR 322 DNA was isolated. Using this procedure, the drug-produced single-strand nick sites which occurred on the strand of DNA which was now labeled with 32P were localized by analysis on denaturing polyacrylamide-DNA sequencing gels. Thus, the sites of damage that occurred when either form I DNA or linear DNA was incubated with drug could be directly compared.

Figure 1 shows an autoradiograph of a sequencing gel containing DNA which had been incubated with drug, in either the linear or the superhelical form, and the densitometric scans of the lanes within the areas labeled A, B, and C are shown in Figure 2a,b. In the scan of the linear DNA control, lane 1 (scan l), several minor peaks are evident, demonstrating the existence of a small amount of cleavage within the untreated DNA fragment. The scan of the superhelical (form I ) DNA control (scan 9) shows that cleavages occurred at sites similar to those in the linear DNA control but were relatively more extensive. [These two control scans were measured at a 40% higher densitometer gain setting than those shown in scans 2 and 3 (Figure 2a) and 7 and 8 (Figure 2b).] It should be noted that following the initial incubation of form I DNA in the absence of drug and subsequent isolation, approximately 10-12% was determined to be converted to the nicked relaxed form I 1 configuration. Therefore, the bands present in lane 9 most likely represent the nicks produced during this incubation and/or during the subsequent isolation procedure. The experimental difference between the control DNA lanes in Figure 1 is that the DNA in lane 9 was incubated in drug reaction conditions (minus drug) as form I DNA, whereas the DNA in lane 1 was incubated under these conditions as the isolated 145-bp restriction fragment.

The amount of cleavage observed in DNA fragments which were obtained from samples incubated in the absence of drug varied from experiment to experiment. However, upon relatively extended exposure times, cleavage was always observed in the autoradiographs.

Scan 3 in Figure 2a shows that cleavage of form I DNA treated with BLM A2 under conditions which produced an average of 0.86 break per DNA molecule occurred at a number of different sites, many of which were also nicked to a relatively

302 BIOCHEMISTRY

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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-bp restriction 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 32P- labeled 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.

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 pas- itions, 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

MIRABELLI, HUANG, A N D C R O O K E

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~ O U R E 2 (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

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 exten- sively 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 V O L . 2 2 , N O . 2, 1 9 8 3 303

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 1 DNA 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 rcdud . 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 <<5.0 breaks per molecule, respectively. In comparing these scan patterns with those in scan 2, which represents the cleavage pattern produced by BLM A, on linear DNA, it is seen that within area A, the cleavage patterns are very similar. The two small peaks in the middle of area A in scan 2 were not resolved in scans 3-5, but were contained in the shoulders of the two larger adjacent peaks. Area B in scans 3-5 contains sites of extensive cleavage while the linear DNA (scan 2) was not significantly cleaved by BLM A2 within this sequence. It can also be seen that the largest peak in this area in scans 3-5 corresponds to a site found to be nicked in the control (scan 1). Although not all the peaks within area B in scans 3-5 of Figure 4a are well resolved, they do correspond to those sites cleaved in area B on form I DNA by BLM A2 as shown in scan 3 of Figure 2a. Within a m C (Figure 4a), t h m is a relatively major peak in scans 3-5 which corresponds to the location of a peak in the control scan (1). However, no signiftcant cleavage occurred at the companding location in linear DNA treated with BLM A, (scan 2). It is also noted that to the left of the major peak in area C in scans 3-5 there is a peak which increased in area relative to the major peak as the amount of degradation of DNA by BLM A2 increased (scans 3-5). This peak corre sponds to the location of a G-C sequence which is cleaved on linear DNA by BLM A, (scan 2, Figures 2a and 4a). Note that the cleavage patterns produced by BLM A, on linear DNA (lane 2) in Figures 1 and 3 are equivalent.

The scans of the lanes in the autoradiograph in Figure 3, corresponding to DNA treated with TLM A, are shown in Figure 4b. Scans 10 and 11 show the cleavage pattern produced when form I DNA was incubated with TLM A under conditions which produced on average 1.2 and X . 0 breaks per DNA molecule, respectively. In a manner similar to that

I 2 3 4 5 6 7 8 9 1 0 1 1 1 2

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FIGURE 3 Sequencing gel analysis of drug-produced oligonuclmtides obiained when either form I or a 145-hp restriction fragment of pBR 322 DNA is used as the substrate (11) (see text for explanation of cxperimntal p d u r e s ) . 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 2, 6-9, and 12 correspond to reactions with the isolated 3’P-labeled restriction fragment. Lanes I , 3-5, IO, and 11 contain DNA which was incubaled as form I DNA in the presence or absence of drug after which the DNA was ’,P labeled and the fragment isolated. (Lane I ) Control (no drug); (lane 2) 200 pM BLM A,; (lane 3) 100 nM BLM A,; (lane 4) 200 nM BLM A,; (lane 5) 400 nM BLM A,; (lane 6 ) T + C (base-specific chemical reactions); (lane 7) C (base-specific chemical reactions); (lane 8) A > C (bas6spcirc chemical reactions); (lane 9) 200 pM TLM A, (lane 10) 200 nM TLM A: (lane I I ) 400 nM TLM A: (lane 12) (no drug) control. All BLM A,, TLM A, and wntrol reactions were done under standard drug reaction wn- ditions (Materials and Methods). Areas labeled A, B. and C are depicted as densitometric scans in Figure 4a,b and correspond to respective areas in Figure 1

obscrved with BLM A,, the sites cleaved within area A by TLM A when form I DNA was used as substrate (scans 10 and 11) were also preferentially cleaved on linear DNA (scan 9). Area B in scans 10 and 11 contains sites which were cleaved when form I DNA was incubated with TLM A, but which were not cleaved when linear DNA was incubated with the drug. Within area C, there are two peaks in scans 10 and 11 which correspond to sites on the DNA which were cleaved when TLM A was incubated with form I DNA (Figure 2b, scan 8). The smaller peak corresponds to a site which was also cleaved when the linear DNA was incubated with TLM A (scan 9). The larger peak in area C in scans 10 and 11 mesponds to a site which was not cleaved on the linear DNA but which was also nicked in the form I DNAs incubated with BLM A, (Figure 4a, scans 3-5) and incubated in the absence of drug (scan 1).

It has been reported that the site of damage in DNA produced by BLM is not a substrate for the enzyme polymerase I (Niwa & Moses, 1981). The following experiments were performed to verify this observation and to ensure that the differenccP observed in these drugs’ DNA-breakage specificities on linear DNA vs. form I DNA were not due to the incor-

304 B I oc H E M I S T R Y M I R A B E L L I , H U A N G , A N D C R O O K E

poration of radioactive label at the sites of drug-produced nicks in form I DNA.

Isolated form I pBR 322 DNA was first restricted with AuaI and isotopically labeled at the two Aud-generated termini of the plasmid with polymerase I (large fragment) and [a- 32P]dCTP. This DNA was incubated in the presence of BLM A2 or TLM A and then digested with MspI, and finally, the labeled 145-bp fragment was isolated (see Materials and Methods). Alternatively, DNA was first restricted with AuaI and then incubated in the presence of drug. This DNA was then labeled and a 145-bp restriction fragment isolated as described above. Sequence gel analyses of these restriction fragments showed that the DNA which was isotopically labeled before incubation with drug produced the same banding pattern as the DNA which was first incubated with the respective drug and then labeled (data not shown). These results indicate that the drug-produced nicks in the DNA were not substrates for polymerase I and that the sequence specificity for breakage within this tract of pBR 322 DNA by the drugs was similar when either the isolated restriction fragment or whole linear plasmid was used as substrate.

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FIGURE 4: (a) Densitometric scans of lanes in Figure 3. Numbers of scans correspond to lane numbers. Areas labeled A, 9, and C correspond to respective areas in Figure 3. (b) Densitometric scans of lanes in Figure 3. Numbers of scans correspond to lane numbers. Areas labeled A, 9, and C correspond to respective areas in Figure 3

Discussion

The sequence specificity of BLM A2 and TLM A induced cleavage of DNA using isolated linear DNA was not identical with that observed on superhelical DNA (Figures 1 and 3). Some of the drug-induced sequence-specific breaks which occurred in linear DNA were also cleaved in superhelical DNA. However, a number of site-specific breaks occurred in the DNA which was in the superhelical configuration when treated with drug that were not observed in linear DNA incubated with drug. The cleavage sites which were peculiar to the superhelical DNA relative to linear DNA treated with BLM A, were also evident in the superhelical DNA treated with TLM A. As the number of breaks produced by BLM A, per molecule of DNA increased, the extent of cleavage of the sequence-specific sites appeared to increase relative to that at the cleavage sites peculiar to form I DNA (Figures 3 and 4a). These results indicate that the specificity of cleavage of DNA by these drugs is influenced in part by the topology of the substrate. This observation is consistent with that reported by Lloyd et al. (1979) in which BLM-preferred reactivity at certain sites on PM2 DNA was influenced by the superhelical nature of form I DNA.

It has previously been shown that the binding of BLM A2 to superhelical ColE1 DNA results in the removal of superhelical turns (Povirk et al., 1979). Therefore, binding of the drug results in removal of negative superhelical turns in re- sponse to unwinding of the Watson-Crick duplex turns. Thus, the cleavage sites which were peculiar to the drug-treated superhelical DNA relative to the drug-treated linear DNA could reflect sites of local distortion or unwinding in the superhelical DNA (but absent in linear DNA) which may be particularly favorable for TLM or BLM to bind to and/or cleave. Such sites may be related to kinks which have been postulated to occur in DNA (Crick & Klug, 1975; Sobell et al., 1977). Sastry & Hallee (1978) have implicated kinks in DNA as possible preferential BLM binding sites. The strong influence of NaCl on BLM binding led these authors to speculate that at low salt concentrations the DNA may be sufficiently destabilized locally with a concomitant decrease in melting temperature with preferential binding of BLM at these denatured sites. Soft modes of DNA excited by thermal fluctuations may bring about distortions (similar conditions as those postulated to produce kinks in DNA) which may be particularly favorable for BLM to interact with the DNA and

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partially intercalate between the bases. Our observation that DNA incubated in reaction mixtures in the absence of drug evidenced relatively small amounts of spontaneous cleavage at sites which were peculiar to the drug-treated superhelical DNA (Figures 1 and 2a,b) indicates that these sites are in- herent to the conformational state of the DNA and are not the exclusive result of the dynamics of the drug-DNA in- teractions. The explanation as to why these sites are sus- ceptible to relatively low levels of cleavage under the conditions of the incubation (minus drug) and/or isolation of the DNA used in these experiments is unclear. It is interesting to note that the sites of the most extensive “spontaneous” cleavage occurred at sites of A-T base pairs. These spontaneous cleavage sites are not in the locations of the S1 nuclease sensitive inverted repeat sequences in pBR 322 reported by Lilley (1980) and described as potential cruciform structures by Panayotatos & Wells (1981).

The results of this series of experiments lead us to propose that as a result of the initial cleavage by TLM A or BLM A2 at these “topologically specific” sites, the DNA would then be transformed into the nicked, relaxed form I1 conformation, at which time cleavage at the sequence-specific sites by the respective drugs would predominate.

In genetic structures of eukaryotic systems, DNA may assume specific conformational states such as superhelical or solenoidal forms in higher ordered structures such as nucleosomes and their polynucleosomal arrays (Finch & Klug, 1976; Germond et al., 1975; Hewish & Burgoyne, 1973; Olins & O h , 1974; Oudet et al., 1975; Renz et al., 1979; Worcel, 1977). These superstructures provide a way of controlling gene activities such as transportation, repair, and replication (Axel et al., 1973). Changes of these structures have been proposed to be associated with certain gene functions and regulations. For example, transcriptionally active genomic structures may be partially unfolded or may be different from transcriptionally inactive structures (Garel & Axel, 1977; Newman, 1979; Piper et al., 1976; Weintraub & Groudine, 1976). A decrease in the number of topological turns was associated with differ- entiation in nucleoids of Friend erythroleukemia cells (Luchnik & Glaser, 1980), and relaxation of supercoiling was related to initiation and elongation of DNA synthesis (Mattern & Painter, 1979).

Therefore, the differences in the degradative activity (Huang et al., 1982) and the site/sequence cleavage specificity of the bleomycins depending on the conformational states of genomic DNA may have different biological consequences. The ability of BLM to affect DNA in deoxyribonucleoproteins has been shown to be dependent on the functional state of DNA (Crooke et al., 1975; Muller & Zahn, 1976). Recently, it has been reported that BLM can preferentially degrade DNA in chromatin with an open or active configuration (Kuo, 1981).

The findings presented in this paper demonstrate the ap- parent sequence specificity of BLM and TLM on nicked relaxed circular and linear DNAs. We have recently reported that a major determinant for location of a BLM- or TLM- produced double-strand break is the production of two closely spaced sequence-specijk single-strand breaks on opposite strands of the DNA (Mirabelli et al., 1982b). The summation of these findings suggests that the functional, conformational, and structural state of the DNA may play an important and interrelated role in the mechanism by which these clincially useful antitumor agents interact with and degrade specific sites in cellular DNA.

Acknowledgments We thank Professor Harris Busch, Chairman, Department

of Pharmacology, Baylor College of Medicine, for his en- couragement as well as Wanda Beattie and Drs. s. Mong and A. W. Prestayko for helpful suggestions and criticisms. We also thank Mary Safrit for excellent typographical assistance.

Registry No. BLM A,, 111 16-31-7; TLM A, 65057-90-1.

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Characterization of Deoxyribonucleic Acid Sequences at the 5’ and 3’ Borders of the 100 Kilobase Pair Ovalbumin Gene Domain? William E. Stumph, Melvyn Baez, Wanda G. Beattie, Ming-Jer Tsai, and Bert W. O’Malley*

ABSTRACT: The ovalbumin and the ovalbumin-related X and Y genes are coordinately expressed in the chicken oviduct and are linked within a 100 kilobase pair (kbp) chromosomal domain of DNA which is preferentially sensitive to DNase I [Lawson, G. M., Knoll, B. J., March, C. J., Woo, S. L. C., Tsai, M.-J., & O’Malley, B. W. (1982) J . Biol. Chem. 257, 1501-1507; Lawson, G. M., Tsai, M.-J., & O’Malley, B. W. (1980) Biochemistry 19, 4403-44111. At each end of this domain, the chromatin undergoes a structural transition such that the DNA becomes relatively more resistant to DNase I . In order to understand the mechanisms underlying this structural transition, we have been studying the characteristics of DNA sequences which are found at the 5‘ and 3‘ borders of the domain. We have discovered that members of a dis- persed repetitive DNA sequence family (termed the CR1 family) are located near or within the transition regions of DNase I sensitivity; in addition, the CR1 sequences found at opposite ends of the domain exist in inverse orientations with

E v i d e n c e has accumulated over the past several years that active gene sequences are preferentially digested by the enzyme DNase I. For example, when compared to the bulk of the chromatin DNA sequences, globin gene sequences are preferentially sensitive to DNase I in erythroid cell nuclei but not in nuclei isolated from other cell types (Weintraub & Groudine, 1976). Similarly, in the chicken oviduct where the ovalbumin gene is expressed, those DNA sequences which code

From the Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. Receioed August 19, 1982. This work was supported by the National Institutes of Health (Grant HD-08188) and the Baylor Center for Reproductive Biology (Grant HD-07495).

0006-2960/83/0422-0306$01.50/0

respect to each other. These observations suggest that the CR1 sequences may possibly play a role in defining the ends of the ovalbumin gene DNase I sensitive domain. A consensus CR1 sequence is presented on the basis of a comparison of four homologous genomic sequences which are members of the CR1 family. We also searched for, but failed to obtain, any evidence that specific DNA rearrangements are involved in the ex- pression of the ovalbumin gene. However, a region at the far 3’ end of the domain, approximately 30 kbp downstream from the ovalbumin gene, is highly enriched for HpaIIIMspT restriction sites compared to most regions of the chicken genome. More importantly, this same region exhibits a pattern of methylation which is highly variable from one chicken tissue to another. In particular, DNA in the oviduct is specifically undermethylated in this region when compared to other tissues. Thus, this DNA at the 3’ border may serve as a control region involved in determining the structural state of the chromatin domain in which the ovalbumin gene family resides.

for ovalbumin are preferentially sensitive to DNase I (Garel & Axel, 1976). Such preferential sensitivity to DNase I is not observed in other chicken tissues where the ovalbumin gene is not expressed. Thus, the DNase I sensitivity of a gene is correlated with the ability of that gene to be expressed in any given cell type [see Lawson et al. (1982) for additional references and examples]. This preferential sensitivity, which is observed for active genes when nuclei are digested with DNase I until about 20% of the DNA is rendered acid soluble, presumably reflects a modification in the packaging or the configuration of the nucleosomes in those regions of the chromatin which contain active genes. The altered chromatin structure and preferential DNase I sensitivity measured in this

0 1983 American Chemical Society

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