[CANCER RESEARCH 51. 3930-3937. August 1. 1991] DNA Sequence Specificity of Doxorubicin-induced Mutational Damage in uvrB~ Escherichia coli1

Russell D. Anderson, Martina L. Veigl, Jeff Baxter, and W. David Sedwick2

Department of Medicine, Case Western Reserve University, and Ireland Cancer Center, University Hospitals of Cleveland, Cleveland, Ohio 44106

ABSTRACT (15-18), and that these DNA rearrangements may be involved in doxorubicin-induced cytotoxicity. In the absence of excision repair, doxorubicin caused a striking (41- The sequence specificity or sequence preferences of doxorub fold) increase in the frequency of large deletion mutations extending from the lac operator (lacO) into the lac represser gene (lad) of Escherichia icin and other anthracycline interactions with DNA have only coli. In contrast, there was only a 2-fold increase in the frequency of been explored in purified systems (19-22). Spectroscopic stud small deletions despite a 3-fold increase in overall mutation frequency. ies show that doxorubicin intercalates into dGdC sequences of The 5'-endpoints of doxorubicin-induced lacO and lad/lacO deletions native DNA (20), whereas fluorescence quenching studies sug occurred at the DNA sequence 5'-pyTAA or 5'-AATpy (where py is gest a general affinity for alternating purine-pyrimidine se pyrmidine) (16%), at runs of purines or pyrimidines (41%) and adjacent quences (21). Footprinting studies demonstrate a marked pref to 5'-dGdC or 5'-dCdG doublets (34%). Ninety % (27 of 30) of the erence for association with dGdC-rich regions of DNA that are doxorubicin-induced deletions involving the region of the lacO palindrome flanked by A:T base pairs (19). More recently, the triplet had 3'-endpoints within the palindrome sequence as compared with 40% sequences 5'-A/TCG and 5'-A/TGC (A/T means A or T oc (4 of 10) spontaneous deletions in an untreated set. Doxorubicin-induced cupy the position) have been identified as preferred binding single base substitutions were highly focused at one site (4 of 6) in the / '' region of lad, in contrast to the spontaneous distribution of point sites for daunorubicin, a structurally related anthracycline (22). mutations, where 16 mutants were recovered at 12 different sites. An Doxorubicin preferentially inhibits the Escherichia coli DNA increased frequency (3-fold) of highly focused base substitutions was also polymerase (polymerase I) at GC-rich sequences (23), whereas observed at 2 sites in the lac operator region (at lacO +6, which is a it inhibits E. coli RNA polymerase preferentially at the sequence transition "hotspot" in the spontaneous spectra of both wild type and 5'-TCA (24). Doxorubicin also creates new preferences for uvrBT organisms and at the adjacent +5 site). Notably, the frequency of DNA cleavage by mammalian topoisomerase II within target 1- and 2-base frameshifts did not increase in the doxorubicin-induced DNA sequences, and this cleavage frequently occurs 3' to spectrum, relative to the spontaneous mutation spectrum. These in vivo adenine bases in the DNA sequence (7, 25). observations in E. coli suggest that in the absence of excision repair, This in vivo study focuses on the DNA sequence specificity doxorubicin causes highly focused deletions and base substitutions. These of doxorubicin-directed DNA damage in E. coli. DNA sequence mutations occur adjacent to DNA sequences identified in previous in level analysis of doxorubicin-induced mutation is an initial vitro studies as preferential sites of doxorubicin binding. approach to suggesting mechanisms by which doxorubicin may damage DNA in vivo. In this study, the lac operator and the INTRODUCTION DNA binding region of the laci3 gene in E. coli were used as The mechanisms by which doxorubicin mediates its relatively mutational targets. Procedures used in this analysis allow re selective antitumor effects are not clear since doxorubicin dam covery of all classes of mutations except +1 frameshifts (26). To more fully unmask DNA level effects, doxorubicin-induced ages numerous cellular components (1). However, several ob mutation in the lad gene was analyzed in uvrB~ DNA excision servations support the premise that DNA is directly and/or repair-deficient E. coli, which have been demonstrated to be indirectly an important molecular target of doxorubicin (2). more sensitive to doxorubicin than excision repair-competent Anthracyclines, such as doxorubicin, intercalate into DNA with consequent DNA unwinding, stiffening, and elongation (3). E. coli (12). DNA base modification via oxidative pathways (4), formation of covalent DNA adducts (5, 6), and interference with topoi- MATERIALS AND METHODS somerase II activity resulting in protein-linked DNA strand breaks (7) are all mechanisms through which anthracyclines Bacterial Strains may damage DNA. Furthermore, these drugs can produce The bacterial strain NR3951 A (pro lac) A (bio, uvrB), ara, thi, lesions in DNA that inhibit DNA synthesis (8-11) and provide trpE-9111 ¥'(prolac) (I°,L8)was kindly provided by Dr. B. Glickman substrate for (12) as well as directly interact with DNA repair of York University, Canada. enzymes (13). Clastogenic events have been reported as in vivo For selection of doxorubicin-induced mutants, 84 separate 1-ml consequences of doxorubicin's effects at the DNA level (14). cultures were initiated with approximately 100 bacteria/ml and grown to 6 x 10* organisms/ml. An aliquot containing approximately 1.25 x Finally, there is evidence that extensive chromosome damage 10s organisms was taken from each culture, the cultures were centri- is caused by doxorubicin and other topoisomerase II antagonists fuged, resuspended in 0.25 ml of minimal media at 5 x 10s organisms/ ml, and incubated for 30 min at 37°C.The bacteria were then exposed Received 8/2/90; accepted 5/14/91. The costs of publication of this article were defrayed in part by the payment to doxorubicin (0.33 mivi) in minimal media for 30 min at 37°C(final of page charges. This article must therefore be hereby marked advertisement in volume, 0.5 ml). After the drug treatment period, cultures were centri- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by a research grant from Glaxo, Inc., to W. fuged, resuspended in 1.0 ml of doxorubicin-dilution buffer (1 mM D. S. and M. L. V.. National Cancer Institute Grant R01-CA52683 awarded to NaHPO4, 1 mivi MgCl2, and 0.05% gelatin, pH 7.4) and centrifuged a W. D. S. and an American Cancer Society Ohio Division Cancer Research second time to remove free doxorubicin. The cultures were then washed Fellowship to R. D. A., Hematology/Oncology Training Grant HL-07147-14. once with 1.0 ml Vogel-Bonner salts and finally centrifuged and resus This work was aided by core facilities made available through the Case Western Reserve University Cancer Research Center Grant (no. P30CA43703). pended in 125 n\ of Vogel-Bonner salts. The drug-treated organisms 2To whom requests for reprints should be addressed, at Ireland Cancer Center Research Laboratories. University Circle Research Center. Building 2. 11001 3The abbreviations used are: lad, lac represser gene; lacO, lac operator; X- Cedar Road, Cleveland. OH 44106. Gal, 5-bromo-4-chloro-3-indolyl-D-galactoside: PCR. polymerase chain reaction. 3930

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1991 American Association for Cancer Research. MUTATIONAL SPECIFICITY OF DOXORUB1CIN IN E. COLI were plated for viability determination on Luria Broth plates and for Amplification and Isolation of Mutant DNA for Sequence Level selection of both lad and lacO mutants on minimal medium plates Analysis containing phenylgalactose as described previously (27). The mutation frequency in these target genes was increased approximately 3- to 5- DNA from approximately 80% of the sequenced mutants in this fold under these drug treatment conditions (see Fig. 1). study was amplified and isolated by recombination into M13. DNA from the remaining mutants was directly prepared from the archived X' stocks by the PCR (35). Collection and Preliminary Analysis of Mutants Recombination into M-13. Procedures for cloning and isolating the A set of 420 lad mutants (5 from each plate) were collected for lad mutations by recombination into M13 for subsequent DNA isola analysis. A sample of each of the mutants was transferred to form a tion and sequencing have been described elsewhere (27, 36). Briefly, gridded pattern on phenylgalactose agar plates. The F' episome, car after transferring the F' episome carrying the lad mutation to a X' rying the lad mutation, was then crossed into a recA" strain (\r) and strain for archiving and storage, mutants were again crossed into an colony-purified, an aliquot was frozen, and new grids were prepared M13 virus recipient stain and subsequently infected with M13 virus. both to probe for mutants frequently isolated in the spontaneous Blue plaques, resulting from the ability of productive virus infections mutationa) spectra (i.e., "hotspots") and to test for dominant comple carrying a defective lac repressor to convert X-gal, were then picked for mentation phenotypes. plaque purification of the virus, preparation of virus stocks, and sub sequent DNA extraction and purification (36). Mutant Analysis by Oligonucleotide Probing Polymerase Chain Reaction. One and one-half-ml Luria Broth cul tures of X' bacteria containing the mutant lad gene were prepared from The procedure of Miller and Barnes (28) was used to analyze all 420 each mutant isolate. These cultures were grown to 2 x 10' organisms/ of the doxorubicin-induced mutants for frequently occurring mutations previously identified in the spontaneous wild type and u\rB~ spectra. ml, the bacteria collected by centrifugation, pellets suspended in 0.5 ml of a buffer made up of 8% sucrose, 50 mMTris (pH 8.0), 50 itiM EDTA, For probing, gridded bacterial colonies were transferred by replica and l % Triton X-100, l mg/ml of lysozyme was added, and the samples plating to Gene Screen (Du Pont-New England Nuclear). Following an overnight incubation at 37°C,the colonies were extracted directly on were incubated on ice for 10 min. The cultures were then boiled for 1 min and centrifuged to remove the bulk of the cellular DNA and the gene screen matrix by successive washes with NaOH and sodium coagulated protein debris. Supernatants were extracted twice with saline citrate buffer. The bacterial DNA of the clones was hybridized phenol and precipitated with alcohol. The samples, containing episome with one of the specific probes, washed extensively, and ultimately DNA, were finally resuspended in Tris-EDTA buffer before subsam incubated for 10 min at an empirically determined temperature that melts mismatched probes from the DNA isolates but allows identically pling for amplification by PCR. PCR amplification of lad in the samples was carried out by an matched probes to remain hybridized. The probes for these experiments asymmetric reaction. A 100-fold excess of oligomer (20 nucleotides in were prepared by end-labeling 16-18-mer oligonucleotides with T4- length) from the 5' end of the sense strand (which initiates at a position polynucleotide kinase. Positive colonies were identified by 58 nucleotides upstream from the first lad amino acid codon) and a autoradiography. second 20-mer (which initiates just 5' to the operator region at the 5'- Oligonucleotide probing was used to identify mutants at the frame- shift "hotspot" (positions 620-631), where the gain or loss of the end of the antisense strand) were used. After 45 cycles of amplification with dNTP and Taq polymerase (United States Biochemical Corp., sequence CTGG from a triple repeat of this sequence in the wild type genotype results in frameshift mutations (29) and at another "hotspot" Cleveland. OH) in a Cetus thermocycler. the DNA was isolated by passage through G50 Sephadex spin columns and precipitated at 4°C resulting in an A:T —»G:Ctransition at the +6 position of the operator with 95% ethanol for over 2 h followed by 3-70% ethanol washes at (27). room temperature. Following resuspension of the pellets, the samples Mutant Analysis for Dominant Complementation Phenotypes were directly sequenced with Sequenase (United States Biochemical). DNA Sequencing. DNA was sequenced by a modification of the We limited our DNA sequence analysis to mutants that displayed a dideoxy DNA sequencing method of Sanger (United States Biochemi dominant complementation phenotype (AT*). These include mutations cal) using [35S]dATP and Sequenase. The i~d region of lad can be within the DNA binding portion of the lac repressor (T'O or within the sequenced using a single primer, which hybridizes to position 247 —» lacO sequence to which the repressor protein binds (26, 30). The i~d 234 and the operator region using a single primer, which hybridizes to segment is an ideal mutational target since it is very sensitive to all lacO position +43 —»+62. classes of mutations including base substitutions leading to amino acid changes (30), deletions, and duplications, as well as all frameshifts RESULTS except +1 frameshifts (26). The remainder of the lac repressor is largely sensitive only to structural alterations and base substitutions resulting Fig. \A demonstrates the increased sensitivity of the uvrB~ in nonsense mutations or affecting codons encoding nonpolar amino strain versus the isogenic wild-type strain, NR3835, to the lethal acids, such as glycine (30). The lacO region also appears to be a focal point for deletion mutations (27). These lacO and ¡~dmutations are effects of doxorubicin. Fig. \B depicts doxorubicin-directed referred to collectively in this paper as NC+ mutations. induction of lad and lacO mutations in these strains. This NC* mutations are identified after a simple transfer of the F' experiment shows that the uvrB~ strain is not only more sen episome, containing a lad/lacO mutation, into the CSH52 strain and sitive to the lethal effects of doxorubicin, but is also more replica-plating of the conjugants onto minimal media plates containing susceptible to doxorubicin-induced mutation. Based on these X-gal. The AT* mutations allow synthesis of fi-galactosidase resulting studies, investigations of the DNA sequence specificity of dox in formation of blue colonies in this background because of their orubicin-induced mutation were carried out in the uvrB~ strain. dominance over lad* residing on the chromosome (31-34). Colonies Mutants were collected at a doxorubicin concentration of 0.33 that do not convert X-gal in the CSH52 strain (white colonies) contain mivi, which caused about 80% lethality and a 3- to 5-fold a mutation within the lad coding sequence for the core domain of the elevation of the mutation frequency after a 30-min treatment lac repressor located approximately between positions 210 and 1109 (NC~ mutants). period. The DNA sequence specificity of doxorubicin-induced mu Based on the results of the complementation and probing studies, the remaining NC* mutants were prepared for DNA sequencing. Since tation was first screened by isolating mutants from 84 separate 5 mutants were originally selected from each culture, a unique A'C+ drug-treated cultures that were each initiated with 100 uvrB~ mutant from over one-half of the original cultures was available for organisms. Table 1 presents the distribution of mutations at several sites that are either "hotspots" or representative of sequence level analysis. 3931

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1991 American Association for Cancer Research. MUTATIONAL SPECIFICITY OF DOXORUBICIN IN £.COLI an 8-fold increase in mutation frequency at a single site, position 100 96 (mutation frequency at position 96; spontaneous, 1.14 x 10~8; doxorubicin-induced, 8.6 x 10~8). This is unlike the spontaneous base substitutions in the i~d region occurring in •a. the uvrB~ strain, which were evenly distributed over 12 sites. > Moreover, base substitution frequencies at 2 positions in the d operator (+5 and +6) were also increased 3-fold by doxorubicin. Table 2 depicts the sequence level analysis of the doxorubicin-

O-_ induced NC+ mutations. Most of the doxorubicin-induced dele tions that were sequenced (32 of 33) and most of the uvrB~ spontaneous deletions (11 of 13) have 3'-endpoints in the lac operator region. However, a closer inspection of these muta 0.1 tions revealed that doxorubicin induced much larger deletions; 10O 20O 300 4OO 50O 66% (21 of 32) extended from lacO into the lad gene versus DOXORUBICIN (Jvl) 9% of the spontaneous Hvr/T operator deletions. This point is depicted graphically in Fig. 2, which shows the tendency for B doxorubicin to promote larger deletions than those seen in the spontaneous «vr/Tspectrum. 80 The 32 deletion mutations inclusive of the lacO gene listed in Table 2 are divided into 3 general categories of 5'-endpoints. Twenty-nine of these 32 5'-deletion endpoints from the doxo- 60 rubicin-treated set comply with one of these suggested cate gories, compared with 4 of 11 among the limited number of 4O sequenced spontaneous uvrB~ deletions (data not shown). Six teen % (5 of 32) of doxorubicin-induced 5'-deletion endpoints I- 20 occurred somewhere in the sequence 5'-pyTAA or 5'-AATpy (where py is pyrimidine). Forty-one % (13 of 32) of the 5' - deletion endpoints were found within or adjacent to runs (4 or more) of purines or pyrimidines. Thirty-four % (11 of 32) of 100 2OO 300 4OO 50O the 5'-endpoints fell into a third category in which the minimum DOXORUBICIN (uM) consensus sequence for a deletion endpoint is dGdC* (A,T or Fig. l . Analysis of the effects of doxorubicin on isogenic wild type and HITA" C)* (G or C), where * represents the possible endpoints. In strains of E. coli. E. coli cultures were grown to 5 x 10s bacteria/ml and treated addition, 4 of the 6 base substitutions in the i~J region occurred with doxorubicin in minimal medium. After the 30-min drug exposure period. the organisms were plated either on LB plates to determine the survival (A) or at position 96, which is a similar sequence consisting of a T on minimal medium agar plates containing phenyl galactose to determine the mutation frequency in the presence of the drug (B). •.wild type strain NR3835: adjacent to a dCdG doublet (see Fig. 5). O. HITA"strain NR3951. Figs. 3 and 4 contrast the spectrum of deletion mutations with 3'-endpoints in the lacO palindrome region that are in different classes, as indicated in the legend to Table 1. Doxo- duced by doxorubicin in uvrB~ E. coli, with the spectra of rubicin-induced mutations are compared with those from a spontaneous mutations in wild-type and uvrB~ isogenic strains, collection of spontaneous mutants in uvrB~ E. coli (kindly and mutations induced by 8-methoxypsoralen plus uvA light in provided by Jennifer Halliday from Dr. Barry Glickman's lab the uvrB~ strain. Figs. 3 and 4 illustrate a potentially important oratory) and with the spontaneous mutation spectrum obtained and unique aspect of these 30 doxorubicin-induced deletion from wild-type bacteria (37). Although doxorubicin increased mutations. Several new 3'-deletion endpoints, identified as overall mutation frequency 3-fold with respect to spontaneously occurring mutations in the uvrB~ strain, the proportion of multiple isolates, were induced by doxorubicin (see Fig. 3). mutations at each "hotspot" is unchanged (see Table 1A). For Most were localized to the stem sequences of the putative example, in both spontaneous and drug-induced uvrB~ spectra, operator stem-loop structure. The distribution of these deletion the percentage of NC* mutations was unchanged; furthermore, endpoints between the putative stem and its unpaired loop in in both cases, the bulk of these /VC+ mutations were T —>C doxorubicin-treated organisms are compared with the distri transitions at the "hotspot" +6 position of the operator. bution of similar deletion endpoints recovered in studies of Sequence analysis of the remaining NC* mutants demon spontaneous spectra in Fig. 4. Ninety % of the doxorubicin- strates the striking differences between the spontaneous muta induced lacO deletions involving the region of the lacO palin tions in the uvrB~ strain and the doxorubicin-induced mutations drome contained a 3'-endpoint focused at the stem or at the (see Table IB). There was a 41-fold increase in large deletions juncture with the loop. This observation stands in contrast to extending from the lac operator into the lad coding region. In the other studies summarized in Fig. 4 (27), and Footnote 4, in contrast, there was minimal effect on the frequency of deletions which more mutation sites were identified in the DNA sequence confined to the operator region or deletions in the first 240 that forms the loop (9 base pairs in length) than in the 2 13- base pairs of the lad coding sequence (ru region). Furthermore, base pair DNA sequence regions that make up the palindrome doxorubicin had little effect on the overall frequency of single and form the stem of the putative stem-loop structure. base mutations in the i~a region. However, these base substitu tions were highly focused (see Table 2 and Fig. 5), resulting in 4J. Halliday and B. W. Glickman, personal communication. 3932

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1991 American Association for Cancer Research. MUTATIONAL SPECIFICITY OF DOXORUBICIN IN E. COLI Table 1 Comparison of spontaneous and doxorubicin-induced mutations A illustrates the results of screening procedures used to initially discern alterations in mutation spectra before proceeding with DNA sequencing analysis. One "hotspot" of lad lies at location 620-630 of the gene and represents two-thirds of the mutations isolated in the spontaneous mutation spectrum of strain NR3835 (37). This mutation consists of the addition or deletion of the sequence CTGG from the usual triple repeat of this sequence in the lad gene. The transition mutation, A:T —»G:C,in the operator region (lacO + 6) is also a "hotspot" in the spontaneous spectrum (37). Mutations within the first 240 base pairs of the lad gene (/"*) encompass the region involved in DNA binding and negatively complement the wild type represser »hencrossed into CSH52 (31-34)—thus allowing selection as blue colonies on X-gal minimal media plates. Other mutations scoring as positive in this cross are lacO mutations, many of which are lacO +6 transitions. These mutations are collectively referred to as iVC* mutants here. This test, therefore, detects general shifts in the spectrum of lad mutation, and localizes these mutations for ready analysis by DNA sequencing. B compares the distribution of the "non-hotspot" NC+ mutants by class (i.e., compares the frequency of base substitutions, deletions, and frameshifts). The deletions are subdivided into 3 categories: those that are contained in the first 240 base pairs of the lad gene (i'' region), those that are contained in the operator region of the lac operon, and those that originate in the lad gene and terminate in the operator region. A. General indicators of mutational specificity B. Comparison of mutation frequency of NC* mutants by class" Mutation Frequency (xlO 8) Ratio dorubicin/ Specific mutation Wild type4 uvrB~f 330 MMdoxorubicin Specific mutation uvrB~c 330 MMdoxorubicin'' xIO'718.5%6.3%52.6%13.0%27025xIO"732.4%16.4%23.7%9.1%21974x10~730.4%17.3%26.4%8.6%420Total MutationfrequencyA'C*+6T-.C+CTGG iVC*frequencyBase substitutions1-240 pairsOperatorDeletions1-240base frameshift-CTGG frameshiftTotal mutants screened20 basepairsOperatorlad —»operator'1 frameshiftsDuplicationsTotaland 2 base se-quenced40.018.33.414.82.311.41.12.31.13596.912.910.871.12.223.745.22.20452.4135124110NC* mutants

°T—>Ctransitions at the "hotspot" lacO +6 position are omitted from B. Spontaneous spectrum of isogenic wild type strain (NR3835) (37). ' Spontaneous spectrum of isogenic uvrB" strain (NR3951).4 d Complete sequence data were not obtained from 5 NC* mutants. One was mapped as a large deletion extending out of the lad coding sequence (data not shown). It has previously been suggested that large deletions in lad inhibit homologous recombination into M13 (27). ' Type of deletion most affected by doxorubicin.

DISCUSSION may cause DNA deletions by promoting the formation or hindering the resolution of "active ends." These are free or Doxorubicin induced mutation more readily in excision re pair-deficient (uvrB~) E. coli and greatly increased the frequency protein-associated discontinuities in single- or double-stranded DNA invoked in virtually all mechanisms that explain DNA of large DNA deletions. As discussed below, the deletion end- deletion (41). Doxorubicin may promote the formation of active points occurred where DNA sequence likely to bind doxorubicin ends directly via oxidative scission of DNA strands (4) or by lies adjacent to sequence unlikely to bind the drug. Doxorubicin activating DNA cutting enzymes such as repair endonucleases increased the frequency of deletions in the palindromic se (12) or topoisomerases (7). Furthermore, inhibition of DNA quence of the lac operator gene control region and redirected synthesis (8-11) or alterations in DNA configuration caused by their endpoints within this region. Doxorubicin also influenced doxorubicin (3) may potentiate erroneous resolution of active frameshift and point mutations. These observations imply an ends generated during replication or repair. important role for the sequence specificity of DNA interaction Approximately one-third of the doxorubicin-induced dele in mechanisms of DNA damage by doxorubicin in vivo. Mutation Frequency in uvrB~ E. coli. The enhanced lethal tions in this study are flanked by direct repeats of 4 or more and mutational effects of doxorubicin in excision repair-defi bases, whereas the rest have no obvious sequence homologies cient E. coli suggest that the drug creates recognition sites for at their endpoints. Direct repeats at deletion endpoints suggest that some doxorubicin-induced deletions may have occurred the uvrABC DNA repair system in vivo by damaging or struc through a "copy-choice" mechanism. Here displaced or slipped turally perturbing DNA. Doxorubicin has already been shown to sensitize DNA to nicking by purified uvrABC exinuclease in DNA strands, which anneal at complementary sequences, act vitro (12). The uvrABC excision repair system repairs a wide as intermediates for fixation of DNA rearrangements through variety of DNA damage including pyrimidine dimers induced normal replicative mechanisms (41, 42). These sequence ho by UV light, DNA cross-links induced by agents such as psor- mologies, however, are not an absolute requirement for all alin and cw-platin, as well as various other DNA adducts. These deletion mechanisms. Topoisomerases, transposases, ligases lesions cause DNA kinking, unwinding, and other structural (41), and other postulated proteins (43) may have the potential changes that appear to be recognition elements for uvrABC to facilitate DNA recombination and DNA strand rejoining enzymes (40). Since doxorubicin can induce DNA helical bend reactions by mechanisms that would not involve initial stabili ing, unwinding, or stiffening (3), this drug has the potential to zation through homologous DNA base pairing reactions. These create determinants for Õ/vr/ÕAC-dependentDNA repair. Thus, mechanisms may account for two-thirds of the doxorubicin- organisms with normal uvrABC repair capacity would be better induced deletions that are not flanked by direct repeats. able to resolve the DNA level effects of doxorubicin than Although DNA gyrase is just one of many enzymes with the organisms that lack this repair system. Consequently, excision- potential to mediate anthracycline-induced deletions, recent repair-deficient uvrB' organisms may suffer more mutations at evidence has highlighted the importance of topoisomerase II persistent lesions that are not quickly resolved by the uvrABC inhibition among the effects of these drugs (7, 17). Other DNA excision repair enzyme system. intercalating agents have been shown to cause DNA strand Deletion Mutations. Doxorubicin treatment resulted in an breaks and mutation via effects on topoisomerase II enzymes overall 5-fold increase in the frequency of DNA deletions (44). In this context, although direct effects of doxorubicin on compared with the spontaneous uvrB~ spectrum. Doxorubicin the bacterial topoisomerase II (DNA gyrase) have not been 3933

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1991 American Association for Cancer Research. MUTATIONAL SPECIFICITY OF DOXORUBIC1N IN E. COLI Table 2 DNA Sequence Analysis of Doxorubicin-induced NC^ Mutations"

No. of independent Mutation Position isolates Surrounding sequence Base substitutions Transitions T:A —C:G 72 TGCCGGTGtCTCTTATC G:C —A:T +5 GTGGAATTgTGAGCGGA

Transversions G:C —C:G 155 GGCGATGgCGGAGCTG T:A —A:T 96 TTCCCGCGtGGTGAACC T:A —G:C 96 TTCCCGCGtGGTGAACC Deletions inclusive of the lacO gene' grouped according to 5'-consensus sequence pyTAA- or AATpy-mediated deletions -63 —+1 AATGTAAG/ifa£rr...64bp...r#rflra/ATTGTGAG 72 bp 1125 —+!/ ACGCAATT/a:af,craa...72bp...r£fg£...154bp...mcar/ACAGG AAA 223 bp 976 —+3/ AAGGGCAA/r:ca£ffff...223bp...íacaaí/T:GTGAGCGG 977 _ +4:

Homopurine- or homopyrimidine-me- diated deletions 57 bp -42 -»+15 TAGGCACC/i-fûAïrt...57bp...c^ara/ACAATTTC 106bp 1098 -.+8 CCGACTGG/aaa^cg... 106bp...Hjçf^a/GCGGATAA 128bp 1096 —+28/ TCCCGACT/ggaa&gcgygc... 128bp...cacaca/GGAAA:CAGCTATG 1101 -.+33: 251 bp 948 —+3 GCTGCAAC/r««c...251bp...r/?gaaf/TGTGAGCG 317 bp 900 -.+21 CATCAAAC/uö?

Frameshifts 2bp 117—118 CAGCCACG(K)TTTCTGCG " Sequence analysis of AT* mutants (excluding those that probed positive for the operator +6 transition). * The position of base substitutions, deletion breakpoints, and duplications are according to the numbering system proposed by Farabaugh (38). c Deletion breakpoints are indicated by a /. For deletions flanked by direct repeats, the / and : bracket the region where the breakpoint may have occurred. d bp. size of deletions in base pairs. ' Mutant's 5'-breakpoint is the +7 position located 5' to lad. ^"Hotspot" in the spontaneous spectra of both wild type and uvrB~ bacteria. documented, a role for this interaction cannot be ruled out. The of 94% of the doxorubicin-induced deletion endpoints. Direct focus of both spontaneous and doxorubicin-induced deletions linkage of topoisomerase II inhibition to mutation involving at the extragenic lac operator palindrome may be consistent DNA recombination has been demonstrated in T4-infected E. with a mechanism involving DNA gyrase. This enzyme has coli where the hotspot for acridine-induced frameshifts was been shown to preferentially form complexes at extragenic located at an acridine-induced cleavable complex site (44). The palindromic sequences that may act as intermediates in DNA DNA gyrase inhibitor, oxolinic acid, has also been implicated recombination (45). DNA gyrase also cuts frequently at 5'- in plasmici recombination (47). None of the deletion endpoints dTdG doublets (46), which were observed within 4 base pairs of the doxorubicin-induced mutations, however, fell within a 3934

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1991 American Association for Cancer Research. MUTATIONAL SPECIFICITY OF DOXORUBICIN IN E. COLI bind doxorubicin. The DNA sequence specificity of 5'- and 3'- deletion endpoints is considered separately because our muta tion selection system confines the 3'-endpoints of mutations uvrB +Doxorubicin deleting the lac operator to the operator region. However, operator deletions with 5'-endpoints throughout the lad gene or operator region may be recovered. Therefore, the 5'-deletion endpoints reflect sequence-specific events within a relatively large target for both doxorubicin binding and deletion forma tion. The 3'-deletion endpoints in the operator region, however, are especially interesting because of the operator's DNA struc tural considerations and because it is a gene control sequence. In this regard, another DNA intercalating agent and topoisom- HODLacO erase II inhibitor, 4'-(9-acridinylamino)methanesulfon-/w-anis- idide, has previously been shown to direct DNA damage to an episomal control region in vivo (49). 5'-Deletion Endpoints. As discussed in "Results," most of the 5'-endponts occur within 3 categories of sites in the DNA. uvrB Spontaneous These endpoints concur with a model in which breaks are formed in regions of low drug binding, adjacent to high drug binding sites. Based on the in vitro studies, the 5'-AATpy or Fig. 2. Schematic representation of deletion mutations from spontaneous and doxorubicin-induced mutational spectra in uvrB~ strains of E. coli. Depicted 5'-pyTAA and purine or pyrimidine runs, which constitute the schematically as a horizontal scale are the relative DNA sequence location of the lad. lacO. and lacZ (fi-galactosidase) coding regions of the lac operon. The lacO deletion endpoints in the first 2 categories (Table 2), would palindrome is illustrated as a stem loop structure to highlight the position of the provide poor doxorubicin binding sites. When located adjacent palindrome. Horizontal lines, approximate sequence positions of each individual to good binding sites, however, they may be altered in helical deletion mutant isolate; adjacent numbers, size of individual deletions in base conformation to become sites for nuclease incision. Interest pairs. Deletions are grouped according to common _V-endpoints. Relative posi ingly, the preferred daunorubicin binding site (i.e., 5'-A/TCG tions of the .V-endpoints within the palindromic sequence are illustrated in the adjacent step loop cartoons. *. a mutation common to both spontaneous and or 5'-A/TGC) (22) flanks 80% of these 5'-deIetion endpoints. doxorubicin-induced deletion mutation spectra. The third 5'-breakpoint motif [dGdC*(A,T or C)*(G or Q] contains the preferential binding site for daunorubicin (22). -5 +1 +5 Here, doxorubicin would be expected to bind preferentially at 5 i-T-G-T-G-T-G-G-A-A-T-T-G-T-G-A-G-C- the dGdC doublet and create an incision site at the adjacent WT - Spontaneous A:T base pair. Therefore, in all 3 cases deletion endpoints occur 3 111 uvrB" - Spontaneous" within the regions of decreased drug binding. These results 1 2 1 1 uvrB' & PUVA3 1 suggest that DNA damage induced by doxorubicin in vivo, by uvrB" t DOX4 whatever mechanism, occurs immediately adjacent to sites where doxorubicin would bind DNA in vitro. 3'-Deletion Endpoints. Doxorubicin both increases the fre quency of deletions involving the lac operator and redirects +15 +20 +25 (with respect to spontaneous mutations) the sites at which 3'- G-G-A-T-A-A-C-A-A-T-T-T-C-A-C-A-C-A-3' deletion endpoints occur within the operator (Figs. 3 and 4). i i l l l ill l i Eight-four % of the doxorubicin-induced 3'-deletion endpoints WT - Spontaneous 1 1 1 uvrB" - Spontaneous 2 2 1 uvrB" & PUVA occurred in the lacO palindromic sequence. Fifty-nine % (19 of 1 32) of the 3'-endpoints cluster by or in one of the 2 AATT uvrB' i DOX 224 3 2

Fig. Ì.Localization of deletion mutations with a .V-endpoint in lacO. The -.G . DNA sequence region encompassing a palindrome in the lac operon spans positions -7 through +27 in the lacl/lacO sequence map (38). The number of individual isolates of ^'-deletion endpoints induced by doxorubicin at various G-T.A-A positions is contrasted with deletion endpoints characterized in previously de Dumber of Isolates G-C scribed spontaneous mutation spectra. Palindromic sequence is in bold face. ' Total mutants, 270 (37);2 total mutants. 219 (J. Halliday and B. W. Glickman, T-A Loop Palindrome personal communication); 'total mutants. 152 (39); 4 total mutants. 420 (this T-Ä Treatment (+7 to+14) data set); * the 5' most possible deletion endpoint is used for deletions with A-T-(«20) («n-A-j Spontaneous flanking direct repeats. uvrB" GT 6 sequence identical to the DNA gyrase consensus sequence ¿•C wt 4 (PyPrT*GNPyNNPy-3') generated with oxolinic acid (48). T-A Total 10 ¿•C Doxorubicin, on the other hand, would be expected to alter the i t DoxorubicinuvrB" H 27 predominant DNA gyrase cleavable complex sites (25). G-C DNA Sequence Specificity of Doxorubicin-induced Deletions. (-io) |.A -T-A-T-G-T G-G-A-A-A Doxorubicin may induce deletions by one or more mechanisms, Fig. 4. Comparison of deletions in (he operator region of the lac operon. The but these appear to act at preferential sites in the DNA se operator palindrome is illustrated in a putative stem-loop configuration. The quence. Comparison of the sequence specificity of doxorubicin- number of 3'-delction endpoints falling within the loop versus palindrome (in cluding the first base outside the palindrome at both ends of the palindrome) are induced deletions and doxorubicin-DNA binding suggests a tabulated for 3 data sets: spontaneous wild type (37); spontaneous uvrB~ (). general rule that deletions are induced where DNA sequences, Halliday and B. \\. Glickman. personal communication); and doxorubicin-treated unlikely to bind doxorubicin, are adjacent to regions likely to uvrB~ E. coli. 3935

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1991 American Association for Cancer Research. MUTATIONAL SPECIFICITY OF DOXORUBIC1N IN E. COLI sequences within the operator palindrome (Fig. 3). Structural Frameshift Mutations. With the exception of frameshift mu tations at the +4/-4 "hotspot," smaller frameshift mutations aspects of this region may play a special role in directing deletion endpoints to the AATT sequences that would also fit were not increased in the doxorubicin-induced mutation spec the first of the 5'-endpoint categories. This sequence is brack trum. Frameshift mutations tend to occur at runs of repeated eted by two G:C base pairs between positions —1and +5, and bases (56, 57), where doxorubicin binding would be unlikely. It AATT is repeated at position +18 to +21, where it is flanked should be noted that +1 frameshifts, observed in vitro (54) with by a G:C base pair at the 5'-side and an A:T pair followed by a intercalators, would not have been recovered using the comple G:C pair on the 3'-side. The sequence AATT bounded by G:C mentation test that defined mutations for sequencing in this base pairs has been shown to form a B DNA structure with an study (26). However, all other small frameshift mutations (in abnormally wide major groove (50). This region in the operator cluding -1, —2and +2) would have been recovered, and only may be especially susceptible to distortion in the presence of one was observed. doxorubicin because it is adjacent to a triple repeated TG Conclusions. Doxorubicin directs the generation of deletion sequence from -7 to -2 and a complementary CA repeated mutations in uvrfT E. coli, with a striking focus of 3'-endpoints sequence from position +23 to +28. These alternating pyrimi- in the palindrome of the lac operator. These results illustrate dine-purine sequences would have a potential to undergo B to the potential of mutational analysis to delineate the DNA Z transition, however they would also be expected to bind sequence specificity of in vivo effects of anthracycline drugs. doxorubicin and therefore strongly stabilize the B DNA config However, the origin of these mutations is still not resolved. The uration (51, 52). DNA structural distortion induced by doxo observed mutations may represent either the result of low rubicin may, in turn, direct mutation through disruption or fidelity DNA polymerization, DNA gyrase effects, or the action of an error-prone repair system at doxorubicin-perturbed DNA alteration of interactions with the various proteins acting at this gene control region. These may include the lac represser, sites. These mechanisms may not be stimulated if DNA pertur RNA polymerase, DNA gyrase, topoisomerase I, DNA polym- bations by doxorubicin are recognized and resolved by uvrABC erase, and various repair enzymes. In this regard, daunorubicin enzymes. Additional experiments will identify mechanisms by has recently been demonstrated to inhibit Hpal restriction which doxorubicin focuses DNA damage at palindromic or endonuclease activity, which is specific for the sequence gene control sequences in bacterial and mammalian cells. The role of doxorubicin in illegitimate DNA recombination will also GTTAAC (53). be addressed. Base Substitutions. Base substitutions at the +6 site of lacO occur frequently in most spontaneous lad mutational spectra (27, 36, 37), and doxorubicin increases the absolute frequency ACKNOWLEDGMENTS of this mutation. Mechanisms proposed for transitions at this We wish to specially thank Jennifer Halliday and Dr. Barry Click- site include increased adenine deamination leading to hypoxan- man for providing the contrasting spectrum of the uvrB~ isogenic strain thine miscoding versus decreased mismatch repair due to re- for general comparison with the doxorubicin-induced spectrum. We pressor binding (27). Possible effects of doxorubicin on these also thank Dr. Glickman for provision of reagents and excellent advice mechanisms have not been defined. In vitro studies show, how throughout the course of this work. ever, that other DNA intercalating agents promote base substi tution. This has been attributed to enhanced polymerase errors REFERENCES via formation of drug:polymerase or drug:polymerase:template complexes (54, 55). Supporting a local effect of doxorubicin at 1. Young, R. C., Ozols, R. F., and Myers, C. E. 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Russell D. Anderson, Martina L. Veigl, Jeff Baxter, et al.

Cancer Res 1991;51:3930-3937.

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