Bleomycin-induced mutagenesis in repackaged lambda phage: base substitution hotspots at the sequence C-G-C-C

Mutation Research, 180 (1987) 1-9 Elsevier MTR 04429

Bleomycin-induced mutagenesis in repackaged lambda phage: base substitution hotspots at the sequence C - G - C - C Lawrence F. Povirk Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.) (Received 9 January 1987) (Revision received 25 February 1987) (Accepted 19 March 1987)

Keywords: Apurinic/apyrimidinic sites; Oxidative mutagens; CI gene.

Summary DNA isolated from lambda phage was treated with bleomycin A2 plus Fe 2÷. The bleomycin-damaged DNA was added to lambda packaging extracts and the resulting phage were grown in SOS-induced E. coil Under these conditions, treatment of the DNA with 0.8 t~M bleomycin reduced the viability 'of the repackaged phage to 3% and increased the frequency of clear-plaque mutants in the progeny by a factor of 16. Bleomycin-induced mutations which mapped to the DNA-binding domain of the cI gene we,re subjected to DNA-sequence analysis. The most frequent events were single-base substitutions at G : C base pairs, nearly all of which occurred at cytosines in the sequence P y - G - C . Cytosines in the third position of the sequence C - G - C - C were particularly susceptible to mutation. At A : T base pairs, mutations were less frequent and were a mixture of single-base substitutions and - 1 frameshifts, occurring primarily at G - T and A - T sequences. Thus, the overall specificity of bleomycin-induced mutations matches that of bleomycin-induced DNA lesions (strand breaks and apyrimidinic sites), which are formed at G r C (particularly P y - G - C ) , G - T and, to a lesser extent, A - T sequences. Furthermore, the frequency of various types of substitutions was consistent with selective incorporation of A and T residues opposite apyrimidinic sites at these sequences. The highly selective nature of bleomycin-induced mutations may explain the lack of mutagenesis by this compound in a number of reversion assays.

Bleomycin is an iron-chelating glycopeptide which is widely used in cancer chemotherapy (Hecht, 1979). By a mechanism involving oxidation-reduction reactions of the chelated iron, bleomycin specifically oxidizes the C-4' position of deoxyribose in DNA, resulting in strand breaks as well as apyrimidinic (and, to a lesser extent Correspondence: Dr. L.F. Povirk, Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.).

apurinic) sites (reviewed by Grollman and Takeshita [1979] and by Povirk [1983]). Bleomycin-induced apyrimidinic sites contain a ketone at the deoxyribose C-4' position (Wu et al., 1983; Rabow et al., 1986), and are thus different in structure from apurinic/apyrimidinic (AP) sites formed in DNA by spontaneous hydrolysis or by the action of repair glycosylases. Attempts to assess the mutagenic potential of bleomycin have yielded confusing results. Bleomycin mutagenesis has been reported in yeast

0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

(Moore, 1978) and in Drosophila (Traut, 1980), although little is known of the molecular nature of the mutations. Most mutagenicity assays using Ames tester strains, particularly those which detect base substitutions, have yielded negative resuits (Benedict et al., 1977; Seino et al., 1978). Some mutagenic response was detected in strain AT102, but most of the mutations were small (3-6 base) deletions (Levin et al., 1984). Bleomycin was found to revert the trpE8 allele in S. typhimurium (Podger and Grigg, 1983) when the cells were grown on a bleomycin-containing plate, but not when treated with the drug in liquid culture. Bleomycin was also mutagenic in the multilocus arabinose-resistance assay (Ruiz-Rubio et al., 1985). In either case, however, the molecular nature of the mutations is uncertain. AP sites have frequently been proposed as common intermediates in mutagenesis by a variety of agents (Schaaper et al., 1983; Foster et al., 1983; Loeb, 1985), and heat-induced AP sites in transfected q~X174 or M13 DNA have been shown to be rather strongly mutagenic, producing primarily base substitutions (Schaaper and Loeb, 1981; Kunkel, 1984). Thus, the apparent findings that bleomycin, which directly induces AP sites, has little or no activity as a base-substitution mutagen, are surprising. In the present study, isolated lambda DNA was treated with bleomycin in vitro under conditions known to induce AP sites and repackaged to form intact phage. Following infection of E. coli, mutations were screened in the lambda cI gene, which is known to be highly sensitive to base substitution. The results indicate significant, but highly selective, production of base substitutions by bleomycin. Materials and methods

Copper-free purified bleomycin A2 was a gift of Dr. William T. Bradner, Bristol Laboratories. Bleomycin was dissolved in distilled water at a concentration of 6.5 mM and stored at -20 ° C. The phage lambda ci857 Indl Oam amp, obtained from Dr. F. Hutchinson, was maintained as a lysogen in E. coli A15 cells. Phage were prepared by heat induction of an exponentially growing culture of the lysogen, followed by precipitation with polyethylene glycol and pelleting of the re-

suspended phage in glycerol gradients. Phage DNA was prepared by pronase digestion, phenol and chloroform extractions (see Maniatis et al. [1982] for details), and dialysis against 10 mM TrisHCI-1 mM EDTA, pH 8 and then against 20 mM Tris-HC1, pH 7.2. For bleomycin treatments, 20/~1 of DNA at a concentration of 125 /~g/ml was placed in a microfuge tube on ice. 5/~1 of bleomycin at various concentrations was added and the solution was stirred 40 times with the Pipetman tip. 5 /~1 of 6 /~M ferrous ammonium sulfate was then added and the solution was again stirred, incubated at 37 °C for 30 min and placed on ice for 10 min. 5 /~1 of this mixture was added to 20/~1 of a freezethaw packaging lysate (prepared as described by Maniatis et al., 1982) which had been allowed to thaw on ice for 25 min, and the solution was mixed by stirring. 10 /~1 of sonicated packaging extract (also thawed for 25 rain) was added and mixed by stirring. The packaging reaction was incubated at 22°C for 1 h and then diluted with with 0.5 ml of 6 mM Tris-HCl-10 mM MgSO4 pH 7.2 (SM) and placed on ice for not more than 40 rain before being diluted and adsorbed to host cells. Precise timing of the reagent additions and the thawing of the extracts was required in order to obtain reproducible results. For preparation of host cells, a culture of A15 sup E44 was grown at 37°C in lambdaT broth (containing 10 g/1 bactotryptone, 5 g/1 NaC1, 0.25 g/1 MgSO4, 2 g/1 maltose, 1 g/1 glucose and 50 mM Tris-HC1, pH 7.5) to an OD600 of 0.3. The cells were pelleted, and then washed and resuspended in SM at an OD60o of 2, and kept on ice. In some cases, 1.5-ml aliquots of cell suspension were spread on a 100-mm diameter tissue culture dish and irradiated with 254-nm ultraviolet light at a dose rate of 2 j/m2-sec. The bleomycin-treated phage were then either directly plated on host cells, or grown for a single lytic cycle in host cells and then plated. For direct plating, treated phage were diluted, added to an equal volume of host cells, preadsorbed at 37 ° C for 30 rain, and then plated on a lambda plate (0.2 ml of the phage-cell suspension plus 3 ml top agar). Phage titer and frequency of clear-plaque mutants was assayed after incubation for 24-36 h at 30 o C (Hutchinson and Stein, 1977). Packaging efficiency in control

samples was 0.5-2 × 108 plaque-forming units per /zg DNA. For lytic growth in culture, 0.1 ml of the phage suspension was added to 0.4 ml of SM and 0.5 ml of host cells, and preadsorbed (37 o C, 30 rain). 0.5 ml of this mixture was added to 25 ml of prewarmed lambdaT broth lacking magnesium and maltose, and grown at 3 7 ° C for 2.5 h. Under these conditions, preadsorbed phage undergo a single lytic cycle but readsorption of progeny phage is prevented by the low magnesium concentration and low cell density. Cells were then removed by centrifugation (4000 g, 10 min), and MgSO 4 (10 raM) and chloroform (2%) were added to the phage suspension. Progeny phage were plated as above using unirradiated plating cells prepared from an overnight A15 culture. Despite the presence of the sup E44 suppressor allele in A15 cells, at least some clam mutants show a clear-plaque phenotype when plated on this strain, even when the amber codon occurs at a glutamine residue (Povirk and Goldberg, 1986; Hutchinson and Wood, 1987). Sequence analysis (Povirk and Goldberg, 1986) employed a modification of the methodology of Skopek and Hutchinson (1984). Briefly, clearplaque mutants were plaque-purified, and c I mutants were selected by a complementation test. The mutations were localized within the cI gene by deletion mapping. Those found to be upstream of base 250 were subcloned into M13mp18 (K. Tindall and F. Hutchinson, personal communication) and sequences were determined by the dideoxy method.

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Results

Effects of bleomycin treatment lambda phage

either plated directly or grown for one lytic cycle in liquid culture. Under these conditions, lambda DNA was highly sensitive to the drug (Fig. 1). Treatment with less than 1 /~M bleomycin reduced dramatically the ability of the DNA to form infective phage when combined with lambda packaging extracts. Fe 2+ alone, at this concentration, had no effect. Ultraviolet-induced activation of the SOS response in host cells had little if any effect on the survival of the phage. The loss in infectivity is probably due to bleomycin-induced double-strand breaks (Povirk et al., 1978) in the lambda DNA, which would prevent it from being packaged. In

on repackaged

In order to avoid possible problems with accessibility of cellular D N A to bleomycin, drug treatments were performed using isolated DNA. Bleomycin was added to lambda DNA and then activated by addition of a slight molar excess of Fe 2÷ (Sausville et al., 1978). The treated DNA was immediately combined with packaging extracts. The resulting phage, containing bleomycindamaged DNA, were adsorbed to exponentially growing SOS-induced or uninduced cells, and

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BLEOMYCIN ( .uM )

Fig. 1. Survival (top) and frequency of clear-plaque mutants (bottom) for repackaged phage containing bleomycin-treated DNA, when directly plated on cells irradiated with 20 J/m 2 of ultraviolet light (z~)or on unirradiated cells (e). Points represent the geometricmeans (survival)or arithmetic mean (mutation rate) and error bars show the range of values obtained in 3-4 separate experiments.The mutation rate at zero bleomycin concentration was 0.005+0.001% with irradiated cells and 0.002_+0.001% with unirradiated cells.

contrast to isolated DNA, intact phage were found to be virtually refractory to bleomycin treatment; even 100 /~M bleomycin in the presence of 100 ~tM Fe 2÷ had no effect (data not shown). Phage containing bleomycin-treated DNA, when directly plated on host cells, showed an increased frequency of clear-plaque mutants. At a dose which reduced the viability of phage to a few percent, mutation rates of 0.08-0.10%, or about 16-20 times background, were obtained in ultraviolet-irradiated cells. The mutation rate in unirradiated cells was about 4-fold lower, suggesting that most of the mutagenesis was SOS-dependent. However, the difference between SOS-induced and uninduced cells was not nearly as great as that Seen with ultraviolet-irradiated phage, which had a 30-fold higher mutation rate in induced cells (data not shown). The SOS-dependence of bleomycininduced mutagenesis is being further investigated. However, preliminary results suggest that there is some mutagenesis even in AB2463 recA- cells, although the frequency is lower than in either uninduced ABl157 cells (an otherwise isogenic strain) or uninduced A15 cells. When treated phage were grown in liquid culture for one lytic cycle, and then the progeny phage were plated, rather higher mutation rates (approximately 0.2-0.3% at survival levels of between 4 and 10%) were obtained than when treated phage were plated directly. The reaon for this increased mutation rate is unknown. For sequencing studies, the direct-plating protocol has the advantage that each mutant isolated is known to be of independent origin. However, in order to determine whether there was a qualitative difference in mutagenesis, mutants derived from both protocols were analyzed.

Mutant sequence analysis In the experiment chosen for sequencing of mutants, phage were treated with 0.8/~M bleomycin and plated directly, resulting in 3% survival with 0.08% clear-plaque mutants in irradiated cells, and 2% survival with 0.02% clear-plaque mutants in unirradiated ceils. An aliquot of the phage was also grown through one lytic cycle with irradiated cells, resulting in a mutation rate of 0.22% in the progeny phage; the background mutation rate with the lytic cycle protocol was 0.016%.

Of the mutants formed in irradiated cells, about half were c I - , and of these, about 60% produced turbid plaques when crossed with the phage KH70 Nam, indicating a mutation upstream of base 250 (Skopek and Hutchinson, 1984). Sequences of 32 of these mutants were determined, 25 from the direct-plating protocol and 7 from the lytic cycle protocol. Sequences were also obtained from 5 additional mutants, isolated from a second experiment using the lytic cycle protocol and SOS-induced cells (bleomycin concentration: 0.6 ~M, survival: 10%, mutation rate: 0.19%). Since all of the 12 lytic cycle mutants were either different or obtained from different experiments, and the remainder were derived from directly plated damaged phage, each of the mutants must have arisen from an independent mutational event. As shown in Table 1, most of the mutations were single-base substitutions at G : C base pairs, primarily G : C ~ A : T transitions and G : C ---, T : A transversions. Substitutions at A : T base pairs, primarily A : T ---, T : A transversions, were less frequent. Several specificities regarding neighboring sequences were apparent (Table 2). Overall, the base directly preceding the substituted pyrimidine base was a G in 24 of 29 mutants, and the base two positions upstream was a C in 22 out of 29 cases. Of the 21 substitutions at G : C base pairs, all but one occurred at a G - C sequence, and 19 occurred at a P y - G - C sequence. Substitutions involving A : T base pairs occurred at both G - T and A - T sequences. As discussed below, these specificities accurately reflect the distribution of bleomycin-induced lesions in DNA that have been previously reported. However, the most striking feature of the spectrum was the concentration of a large proportion of the mutants at a few specific sites in cI. Although there are at least 120 potential mutational targets in cI at which a base substitution will give a clear-plaque phenotype, two-thirds (20/29) of the bleomycin-induced base substitutions occurred at 5 sites. In most cases, 2 or 3 different types of substitutions were detected at these single sites. The three G : C hotspots, which accounted for nearly half of the single-base substitutions, shared a common four base sequence, C - G - C - C , the substitutions occurring at the third position. These are the only three C - G - C - C sites in bases 1-250

TABLE 1 BLEOMYCIN-INDUCED GENE a

MUTATIONS

IN

THE

cI

Single-base substitutions Change

Occurrences

Positions in Gene b

A :T ~ T :A

6

1, 38 c, 143, 143, 143, 310 d

A:T--* G : C G:C~A:T

2 9

G:C~T:A

9

G : C --' C : G

3

1,! 5, 61 c, 113, 113, 140, 169, 188, 188, 245 ~ 45, 140, 140, 148, 188, 188 c, 245, 245,245 188, 245, 245

Frameshifts Type

Occurrences

Positions in gene

-1 G:C -1 A:T + 1 A :T

1 5 1

(160-161) e - 4 2 , (9-14) e, 48, 134 ¢, 163 (71-77) e

Other G : C ---, T : A at base 45 plus - 1 A : T frameshift at base 48 a For the complete cI sequence, see Sauer (1975). b Underlined mutants are those isolated using the lytic cycle protocol. c Isolated in a second experiment, using the lytic cycle protocol; all other mutants were isolated in a single experiment. d Mutations with base substitutions downstream of base 250 occasionally give a positive cross with the KH70 mapping phage due to the absence of the ci857 mutation in KH70 (Hutchinson and Wood, 1987). e Due to the presence of adjacent identical bases, the exact position of the frameshift is indeterminate.

of cI where a change at the third position will lead to an amino acid change. A small number of frameshifts were also detected, primarily - 1 frameshifts at A : T base pairs. Two frameshifts occurred in runs of adjacent T bases; these may have been unrelated to bleomycin-induced damage as this type of mutation is frequently produced when SOS-induced cells are infected with undamaged phage (Wood and Hutchinson, 1984). Of the frameshifts that occurred at lone A : T base pairs, two were found at a G - T sequence (positions - 4 2 and 48), one at an A - T (position 163) and one at a C - T (position 134). Positions - 4 2 and 48 share the sequence C-G-T-G-C, and both share the sequence C - G - T with the substitution hotspot at base 143.

The sequence at the - 1 G : C frameshift is T - G - C - C . Thus, although only a few - 1 frameshifts were recovered, there is some suggestion that the sequence specificity is similar to that of the base substitutions. There were no obvious differences between the mutants recovered from the direct plating and lytic cycle protocols. Three of the lytic cycle mutants were identical to mutants recovered from direct plating. Although an attempt was made to sequence mutants from an aliquot of the bleomycin-damaged phage which was plated on uninduced cells, only 3 of 20 mutants which were isolated contained a sequence change in cI upstream of base 250. These were A : T - - , T : A transversions at positions 38 ( G - C - T - T ) and 143 ( C - G - T - T ) (both identical to mutants isolated from SOS-induced cells) and a G : C ~ A : T transition at position - 3 9 ( C - A - C - C ) . Thus, base substitutions are also generated in cells which have not been SOS-induced, but the sample size is too small to indicate whether the specificity has changed. As in any system which selects mutants on the basis of gene function, the spectrum of cI mutants isolated in this study reflects both the specificity of the mutagen and the nature of the mutable sites in the target gene. The density of detectable base substitutions in bases 1-250 of cI is unusually high, despite the fact that some amber mutants may go undetected in the suppressor host cells. In fact, substitutions have been found at nearly half the bases in the sequence (Hutchinson and Wood, 1987). Therefore the cI gene is ideally suited for the study of mutagens such as bleomycin, which have complex sequence specificities. Nevertheless, the possibility remains that there may be other sequences which are hotspots for bleomycin-induced mutagenesis but which do not occur at mutable sites in cI. On the other hand, it should be kept in mind that base substitutions will normally be overrepresented in a cI mutational spectrum, compared to spectra that would be obtained in most other genes. Discussion

The results of the present study clearly indicate significant production of single-base substitutions

by bleomycin in repackaged lambda phage. The spectrum of mutations obtained is markedly different from that seen with any other agent, including other oxidative mutagens with free radicalbased mechanisms, such as ,/-rays (K. Tindall and F. Hutchinson, personal communication) and neocarzinostatin (Povirk and Goldberg, 1986). It is also markedly different from the spectrum of spontaneous mutations seen with untreated phage grown in either SOS-induced or uninduced cells (Wood and Hutchinson, 1984; Skopek and Hutchinson, 1984). Two distinct types of DNA lesions are known to be produced by bleomycin, in roughly equal amounts. Bleomycin-induced direct strand breaks involve oxidative cleavage of the (C-3')-(C-4') bond in deoxyribose. A base attached to a 3-carbon sugar fragment (base-CH=CH-CHO) is released, leaving a break with a two-carbon fragment (glycolic acid) on the 3' end and a phosphate on the 5' end (Giloni et al., 1981). Bleomycin-induced AP sites involve oxidation of C-4', release of the free base and opening of the sugar ring. The resulting AP site contains an aldehyde at C-1' and (unlike AP sites in heat-depurinated DNA) a ketone at C-4' (Wu et al., 1983) The phosphodiester back-

TABLE 2 SEQUENCES S U R R O U N D I N G SITES OF BLEOMYCINI N D U C E D BASE SUBSTITUTIONS Position

Number of mutants

Sequence a

245 188 140 113 5 45 61 148 169

6 * 5 * 3* 2 1 1 1 1 1

CGCC CGCC CGCC CGCA TGCT GACG TGCT A G CA TG CA

143 1 38 310

3 3* 1 1

CGTT CATA GCTT AGTA

Sequence surrounding the pyrimidine in the mutated base pair (italicized) is given in the 5' to 3' direction. * More than one type of substitution detected at these sites (see Table 1).

a

TABLE3 C O M P A R I S O N OF S E Q U E N C E SPECIFICITIES OF B L E O M Y C I N - I N D U C E D MUTAGENESIS A N D DNA CLEAVAGE -

Mutation a Cleavage b Cleavage c

2 Position

-

1

Position

C

T

A

G

C

T

A

G

22 7 5

3 7 6

2 2 1

2 1 3

1 0 1

0 0 2

4 0 5

24 17 7

Number of mutants in which the indicated bases were found either one or two positions to the 5' side of the pyrimidine in the mutated base pair. Only the - 1 and - 2 positions are shown since no correlations were seen at other neighboring positions. b Number of "extensively cleaved" sites in 5 restriction fragments in which the indicated bases were found on the 5' side of the pyrimidine at which cleavage occurred. In this study, heat denaturation was employed; thus, the specificity may reflect direct strand breaks only (from Mirabelli et al., 1982b). Same as b, except all cleavage sites (both weak and strong) found in a single restriction fragment are included. Since alkali was used to denature the D N A for sequence analysis, the specificity presumably reflects both direct breaks and apyrimidinic sites (from Takeshita et al., 1978).

bone remains intact, but will form a strand break upon exposure to alkali. No bleomycin-induced DNA base damage has been reported. Although there have been many studies of the sequence specificity of bleomycin-induced DNA damage in vitro, none have carefully distinguished between direct breaks and alkali-labile AP sites. Studies in which DNA was denatured in hot alkali before analysis on sequencing gels presumably detect both strand breaks and AP sites (D'Andrea and Haseltine, 1978; Takeshita et al., 1978, 1981), while those employing heat denaturation alone may detect only direct breaks (Mirabelli et al., 1982a, b). All available studies, however, are in agreement that the predominant sites of bleomycin attack are pyrimidines in the sequences G - C and G-T. Takeshita et al. (1978) also reported cleavage at several A - T sequences. In addition, two studies showed that the nucleotide residue two bases preceeding the residue attacked was usually a pyrimidine (Table 3). Despite differences in detail, both these specificities are clearly reflected in the spectrum of bleomycin-induced base

substitutions in cI, strongly suggesting that the mutations result from misreplication of DNA at sites of bleomycin-induced damage. The specific targeting of the sequence C - G - C - C , however, is not predicted by the in vitro damage, which shows little specificity with regard to bases on the 3' side of the sites of bleomycin attack. Thus, the existence of these hotspots may reflect a second level of specificity related to repair or replication. The simple G:C-richness of the hotspots could be a contributing factor; overall, the region of cI in which mutants were sequenced is relatively A : T rich. The mutational spectrum alone gives no direct indication which of the two known bleomycin-induced lesions, strand breaks or apyrimidinic sites, are responsible for mutagenesis. However, the apyrimidinic site appears to be a more plausible candidate for a mutagenic lesion. Heat-induced apurinic sites, in both single-stranded bacteriophage DNA and double-stranded plasmid DNA, clearly have a strong mutagenic potential (Schaaper and Loeb, 1981; Bichara and Fuchs, 1986). The mutations recovered from depurination mutagenesis are similar to the bleomycin-induced mutations in that most are SOS-dependent singlebase substitutions, with a minor component of - 1 frameshifts. Furthermore, the distribution of various substitutions is consistent with selective incorporation of nucleotides, in the order A > T > G >> C, opposite the putative apurinic sites (Kunkel, 1983). A similar selectivity is evident in the bleomycin-induced substitutions in that the "new" base pair in the mutant sequence is an A : T in 24 of 29 cases. This selectivity might also explain in part the low rate of mutation at A : T base pairs, since incorporation of an A residue opposite a bleomycin-induced apyrimidinic site at a T residue would not result in a mutation. Thus, the mutational data with both depurination and bleomycin treatment are consistent with a model (Sagher and Strauss, 1983; Loeb, 1985) wherein normal replicative systhesis is blocked at the AP sites, but in the presence of SOS functions, can proceed past the block, usually with incorporation of an A or T residue opposite the AP site. While bleomycin-induced strand breaks cannot be excluded as possible mutagenic lesions, there is no such obvious model to explain how strand breaks

would result in base substitutions. The minor component o f - 1 frameshifts seen with both bleomycin and heat depurination may result from replication which skips the AP site entirely, incorporating a nucleotide complementary to the next nucleotide in the template strand. Such a process might be facilitated by folding of the AP site outside the DNA helix to permit staking of the two adjacent bases. The highly selective nature of bleomycin-induced substitutions may explain, at least in part, the negative results obtained with bleomycin in certain reversion assays (Benedict et al., 1977; Seino et al., 1978). A survey of the standard Ames tester strains used to detect base substitutions (Hartman et al., 1986) reveals that none of them have a G - C as the target sequence, and only one allele, hisG428, has a G-T. This allele was reverted by bleomycin, but only when present on a multicopy plasmid; furthermore, most (76%) of the revertants were 3- or 6-base deletions of the ochre codon (Levin et al., 1984). Nevertheless, the remaining revertants (which were uncharacterized) may represent substitutions at the G - T sequence. The failure of bleomycin (as well as ionizing radiation [K. Tindall, personal communication]) to induce any similar small deletions in cI suggests a fundamental difference in mutational mechanism in the two systems, possibly related to the presence of multiple copies of hisG428 in the AT102 tester strain. Another possible factor contributing to the low mutagenic activity of bleomycin in bacteria may be limited accessibility of cellular DNA to the drug. Studies by Yamamoto and Hutchinson (1984) with E. coli indicated that cells were killed by concentrations of bleomycin which produced surprisingly little DNA strand breakage. These authors proposed that the results could be explained by the localization of DNA damage to a small region of the bacterial chromosome, sucti as membrane-associated DNA. Such localized DNA damage might be lethal but essentially nonmutagenic. Finally, it should be noted that, even in lambda phage, mutation rates seen with bleomycin are significantly lower than those seen with an equitoxic dose of potent SOS-dependent mutagens such as ultraviolet light (Wood et al., 1984) and neocarzinostatin (Povirk and Goldberg, 1986). In

the larger g e n o m e of whole cells, the toxicity of b l e o m y c i n may obscure mutagenesis entirely. I n m a m m a l i a n cells, b l e o m y c i n - i n d u c e d m u t a genesis was f o u n d to be similar to that i n d u c e d by X-rays in that a m u c h higher forward m u t a t i o n rate was detected at the gpt locus in AS52 cells t h a n at the hprt locus in C H O - K 1 - B H 4 cells. This p a t t e r n m a y be characteristic of agents which produce p r e d o m i n a n t l y large deletion m u t a t i o n s (Stankowski et al., 1986). However, the data of the present study suggest that, even if b l e o m y c i n is only a weak base s u b s t i t u t i o n m u t a g e n , it could have i m p o r t a n t effects on genes in which a bleomycin-sensitive sequence is present in a critical part of the gene. F o r example, the sequence C - G - C - C , a hotspot for b l e o m y c i n - i n d u c e d m u tation in E. coli, is present in the c o d o n 12 region of the h u m a n c-Ha-rasl protooncogene, a n d a n y s u b s t i t u t i o n at the third nucleotide of the sequence will activate the gene (Seeburg et al., 1984). Thus, if the m u t a g e n i c specificities of b l e o m y c i n in bacterial a n d m a m m a l i a n cells are similar, the drug may be an efficient activator of c-Ha-rasl. A t t e m p t s to test this prediction are in progress.

Acknowledgements I t h a n k Kristie Penley a n d J o n a t h a n B o w m a n for technical assistance, Dr. Eric W e s t i n for providing sonicated packaging extracts, Dr. W i l l i a m T. Bradner a n d Bristol laboratories for s u p p l y i n g bleomycin, Dr. F r a n k l i n H u t c h i n s o n for assistance in setting up procedures for sequencing cI m u t a n t s , a n d Dr. J o h n Schuetz for a critical reading of the manuscript. This work was s u p p o r t e d by U S P H S G r a n t CA40615 a n d an award from the G r a n t s - i n - A i d P r o g r a m for F a c u l t y of Virginia C o m m o n w e a l t h University.

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