Mutational Specificity of Depurination (Bacteriophage M13mp2/Transversion Mutagenesis/SOS Repair/Base Selection/Noncoding Lesion) THOMAS A

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1494-1498, March 1984 Genetics

Mutational specificity of depurination (bacteriophage M13mp2/transversion mutagenesis/SOS repair/base selection/noncoding lesion) THOMAS A. KUNKEL Laboratory of Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 Communicated by James A. Miller, November 16, 1983

ABSTRACT The mutagenic consequences of damage to insertion of dAMP at apurinic sites (21), leading to distinc- DNA produced by low pH and high temperature have been tive G-C -+ T-A and A-T -- T-A transversions. determined in a forward mutational system capable of detect- The use of a single-base-substitution assay involving re- ing all classes of mutagenic events. When damaged single- version of nonsense codons in an essential gene focuses on a stranded DNA from bacteriophage M13mp2 is used to trans- very specific set of base changes at a limited number of sites. fect competent Escherichia coli cells, a 15-fold increase in mu- The inability of this system to detect all possible single-base tation frequency, measured as loss of a-complementation by changes as well as other classes of mutagenic events has led the lac DNA in the phage, is observed compared with an un- to the development of a forward mutational assay in a nones- treated DNA control transfection. The enhanced mutagenicity sential gene. This assay selects for loss of f3-galactosidase is largely dependent on induction of the error-prone SOS re- "a-complementation," encoded in the DNA of bacteri- sponse and is proportional to the number of lethal hits intro- ophage M13mp2 developed by Messing et al. (22). Because duced into the DNA. The effect is abolished by treatment of selection is for loss of a nonessential gene function, a wide the damaged DNA before transfection with either apurinic/ spectrum of mutagenic events can be scored. The precise apyrimidinic endonuclease or alkali. Based on these observa- nature of the mutagenic events, whether base substitutions, tions and the rate constants for formation of the known heat/ frameshifts, deletions, additions, or rearrangements, can be acid-produced lesions in DNA, it is concluded that the major- determined by determining the sequences of the DNAs of ity of the induced mutagenesis results from apurinic sites. the mutants. This system is used here to determine the muta- DNA sequence analysis of 87 spontaneous and 124 induced tional consequences of a noncoding lesion, the apurinic/ mutants indicates that the major effect is on single base-substi- apyrimidinic site. Of particular interest are two issues, the tution mutagenesis with a small increase in (deletion) frame- importance of base-substitution mutations relative to other shift frequency. Approximately 80% of the base-substitution mutagenic events and the specificity of the induced base mutations occur at purine positions in the viral strand, consis- changes in a system capable of monitoring a wide spectrum tent with depurination as the predominant premutagenic le- of base changes at many sites. sion. The preference of guanine over adenine sites mutated is consistent with the preference for depurination of guanine MATERIALS AND METHODS over adenine. Transversions are observed for 57 of 79 (72%) Bacteria and Bacteriophage. E. coli strains S90C [A(pro- induced base substitutions, with a strong preference for inser- lac), ara-, thi-, strA], S90C recA56, NR8036 [A(pro-lac), tion of adenine residues opposite the putative apurinic site. ara-, thi-, trpE9777], and NR8037 [A(pro-lac), ara-, thi-, These data in a forward mutational system provide insight into trpE9777, umu C36::tnS] were provided by R. Dunn and B. the mechanisms used by a cell to replicate DNA containing Glickman of this Institute. E. coli strain CSH50 [ara-, thi-, noncoding lesions. A(pro-lac)/F'traD36, proAB, lacJPZAM15] (the AM15 deletion spans 93 bases of the lacZ gene carried on the episome; Insight into the effects of damage to genetic information re- see Fig. 1), and bacteriophage M13mp2 were obtained from quires analysis of the interactions of cellular replication and J. E. LeClerc (Univ. of Rochester, Rochester, NY). repair processes with specific lesions in DNA. One such le- DNA Preparation. Single-stranded M13mp2 viral DNA sion that has received considerable attention in recent years was incubated at 70°C for various times in 30 mM potassium is the abasic site (1, 2) produced by hydrolysis of the N-gly- chloride/10 mM sodium citrate, pH 5.0. These conditions cosyl bond between the sugar and base (3-5). This base loss introduced one AP site per molecule in 4 min, measured by can occur spontaneously at high frequency (3, 6) and this survival (18). Hydrolysis of AP sites was carried out either frequency can be increased either by base modifications re- by incubating 10 ,ug of depurinated DNA for 30 min at 37°C sulting from certain DNA damaging agents (7-11) or enzy- with 20 units of apurinic/apyrimidinic endonuclease from matically by the action of DNA glycosylases (1, 12). Apurinic/ HeLa cells [fraction VII (23)] (kindly provided by D. Mos- apyrimidinic (AP) sites are quite stable (4, 13), and cells have baugh and S. Linn, Univ. of California, Berkeley, CA) in 500 evolved mechanisms to repair these lesions (12, 14). Unre- ,ul of 50 mM Tris-HCl, pH 7.5/10 mM MgCl2 or by incuba- paired AP sites have been shown to have two biological con- tion in 0.1 M NaOH (final pH 12.8) for 30 min at 37°C. sequences, lethality (15-18) and base-substitution errors SOS Induction. The various strains of E. coli were grown (18). These errors can be produced in vitro with purified at 37°C to a cell density of 2-4 x 108 cells/ml in YT medium DNA polymerases (19, 20) or can occur in vivo in Escherich- (8 g of tryptone, 5 g of yeast extract, 5 g of NaCl per liter, pH ia coli under conditions in which the SOS response has been 7.8), harvested by centrifugation, and then suspended in induced (11, 18, 21). These results, obtained in an assay spe- 0.9% NaCl at 10% of original volume. These cells were irra- cifically designed to detect base-substitution errors that re- diated at 25-150 J/m2 in thin layers in plastic Petri dishes vert 4X174 amber mutations, indicate that for a limited num- with constant gentle shaking. After removal of small aliquots ber of sites in 4X174 DNA there is a strong preference for for determination of cell survival, cells were diluted to 50% of their original volume in prewarmed YT medium and incu- The publication costs of this article were defrayed in part by page charge bated in the dark for 45 min with vigorous aeration. These payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: AP, apurinic/apyrimidinic.

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cells, or unirradiated cells grown to 2-4 x 108/ml in YT me- Table 1. Mutation frequency of heat/acid-treated M13mp2 DNA dium, were treated with CaCl2 to produce competent cells as in normal and SOS-induced cells described (24). Mutation Relative Transfection and Plating. Transfections were carried out UV dose, Lethal hits per frequency mutation by diluting the DNA into 75 mM CaCl2 and adding 2 vol of J/m2 DNA molecule (x 10-4) frequency competent cells. The volume used was determined by the ratio of DNA molecules to cells, which was kept constant for 0 0.0 6.2 ± 1.4 1.0 all variables in a single experiment, typically at 20:1. The 0 3.2 13.6 ± 3.0 2.2 efficiency of transfection was 2 x 10-7 (50 plaques per ng of 100 0.0 11.8 ± 4.0 1.9 single-stranded DNA) and varied 2- to 5-fold over several 100 3.2 97.8 ± 25.3 15.8 experiments. After a 40 min incubation at 0WC, the mixture The transfections, using E. coli CSH50 F' lacZAM15, used either was heat shocked at 420C for 3 min (or 5 min for volumes undamaged DNA (0.5-2.0 ,ug) or heat/acid-treated DNA (5.0-20 larger than 5 ml), then placed at 0C. An amount of this mix- Ag). Values shown are mean ± SD of six independent experiments, ture sufficient to yield 100-500 infective centers per plate all carried out with a single M13mp2 viral DNA preparation. Surviv- was added to 3 ml of0.8% agar in 0.9%o NaCl containing 2 mg al of the UV-irradiated cells was 55 ± 12%. of 5-bromo-4-chloro-3-indolyl ,8-D-thiogalactoside and 0.2 ml of a logarithmic phase culture of E. coli cells (CSH50F' then treatments that eliminate AP sites should reduce or lacZAM15). This mixture was poured onto plates containing eliminate the effect. As shown in Table 2, two independent AP enhanced 30 ml of minimal medium solidified with 1.5% agar and con- methods of hydrolyzing sites reduced the mutagenesis. Hydrolysis by the highly specific (23) HeLa cell taining 0.24 mg of isopropylthio-,3-D-galactoside per plate. These plating conditions were specifically chosen to give in- AP endonuclease (experiment 1) eliminated 95% of the en- tense blue color for wild-type M13mp2 plaques and to allow hanced mutagenicity while alkaline hydrolysis (4) reversed optimum visualization of mutant phenotypes. the effect by 85% (experiment 2). The residual mutagenesis Scoring Mutants and Determination of DNA Sequences. In- above background may represent incomplete hydrolysis of AP sites or the of other lesions activation of a-complementation (25) resulting from a muta- presence (3, 6, 10, 12, 28, 29). tion in the lac DNA in M13mp2 will give rise to mutants These data clearly indicate that the majority of the enhanced readily distinguished as lighter blue or colorless plaques (26). mutagenesis results from apurinic (and possibly apyrimi- Mutants were scored after 12-15 hr of incubation at 370C dinic) sites in the DNA. followed by 24 hr of incubation at room temperature. To SOS Dependence. The mutagenic response was examined eliminate false positives due to plating artifacts, mutant in recMA and umuC- host cells, which are defective for plaques were picked from the plate, diluted in 50 mM sodium SOS-dependent mutagenesis (30, 31). The results (data not borate buffer (pH 9.0), and mixed with an equal dilution of shown) indicate no damage-dependent increase in mutation wild-type M13mp2 phage. Plating this mixture allows a visu- frequency either for the recA or umuC- mutant strains, us- al comparison of phenotypes on the same plate and reduces ing conditions that gave enhanced frequencies for the wild- uncertainty in identifying light blue plaques. Single-stranded type parent strains (32-fold and 11-fold, respectively). These DNA was prepared from the mutants scored in each of three data confirm the SOS dependence of the enhanced mutagenesis and further the independent experiments and sequences were determined by support concept that apurinic sites, the chain-terminator method (27). Two individual oligonu- which are known to impede DNA synthesis (21, 32-34), are cleotides were the responsible lesion. used to prime the reactions for sequence anal- DNA yses, (i) the 15-base primer from Bethesda Research Labora- Sequence Analysis. A total of 87 spontaneous and 124 tories that is to the induced mutants were subjected to DNA sequence analy- complementary coding sequence for ami- sis. The classes of mutations observed are summarized in no acids 11-16 of the lacZ gene and (ii) a newly synthesized 15-base primer (New England BioLabs) complementary to Table 3. The most frequent events under either condition are the coding sequence for amino acids 46-50 of the lacZ gene. Table 2. Reversibility of induced mutagenesis by previous RESULTS treatment of DNA with AP endonuclease or alkali The biological activity of M13mp2 viral DNA, when trans- Mutation Relative fected into competent E. coli cells, decreases in proportion UV dose, Lethal hits Previous frequency mutation to the time of incubation at 70°C in pH 5.0 buffer (data not J/m2 permolecule treatment (x 10-4) frequency shown). This result is similar to that observed with kX174 Exp. 1: AP endonuclease treatment single-stranded DNA (18). It is consistent with the conclu- 0 0.0 - 5.4 1.0 sion that, when using single-stranded DNA subject to few or 0 3.0 - 11.8 2.2 no repair processes, a major biological consequence of de- 0 3.0 + 14.2 2.6 purination is lethality (14-16). Concomitant with this inacti- 100 0.0 - 6.0 1.1 vation is an increase in mutation frequency (Table 1). The 100 3.0 - 57.8 10.7 increase is double that of an untreated DNA control when 100 3.0 + 8.8 1.6 the transfection is carried out using normal competent cells. Exp. 2: alkali treatment However, if the transfection is carried out using competent 0 0.0 - 6.1 1.0 cells made from bacteria UV irradiated to induce the SOS 0 3.0 - 16.6 2.7 response, the mutation frequency increases to 97.8 x 10-4, 0 3.0 + 17.7 2.9 15 times that of the untreated DNA in unirradiated cells. 100 0.0 - 13.8 2.3 Both the actual mutation frequency and the relative increase 100 3.0 - 101.0 16.6 above background are a function of the number of lethal hits 100 3.0 + 27.1 4.4 in the DNA and the UV dose to the bacteria (data not E. coli CSH50 F' lacZAM15 were used for transfections. Cell sur- shown). These phenomena are similar to previous observa- vivals were 61% and 70%, respectively, for experiments 1 and 2. tions using heat/acid-damaged 4X174 DNA (18) and imply Phage survival was unaffected by AP endonuclease treatment and that mutants result primarily from SOS-dependent bypass of was halved by alkali treatment, in both normal and irradiated cells. abasic sites. Percentage reversal was calculated as follows: experiment 1, 1 - Mutagenesis Due to AP Sites. If apurinic (or apyrimidinic) [(8.8-6.0)/(57.8-6.0)] x 100% = 95%; experiment 2, 1 - [(27.0- sites are indeed responsible for the enhanced mutagenesis, 13.8)/(101.0-13.8)] x 100% = 85%. Downloaded by guest on September 24, 2021 1496 Genetics: Kunkel Proc. Natl. Acad. Sci. USA 81 (1984)

Table 3. Frequency of various classes of observed mutants No treatment Treated DNA, induced cells (6.2 x 10-4) (97.80 x 10-4) Events MF Events MF Type of event No. % (X 10-4) No. t (X 10-4) Treated/normal Base substitution 28/87 32.2 (42.4) 2.0 79/124 63.7 (85.9) 62.3 31.3 Double mutation 0/87 <1.2 (<1.5) <0.1 3/124 2.4 (3.3) 2.3 >23.0 (-) Frameshift 9/87 10.3 (13.6) 0.6 7/124 5.6 (7.6) 5.5 9.2 (+) Frameshift 3/87 3.4 (4.5) 0.2 0/124 <0.8 (<1.1) <0.8 <4.0 Deletion 93-base 21/87 24.1 (0) 1.5 32/124 25.8 (0) 25.2 16.8 Other 26/87 29.9 (39.4) 1.9 3/124 2.4 (3.3) 2.3 1.2 When the value for single-base errors (60 x 10-4) is used, it is assumed that there are 3.2 AP sites in a molecule containing 3600 purines, and the fact that (at least) 35 sites in the target are mutable (Fig. 1) is considered, the bypass frequency, calculated as described (18), is estimated to be 20%o. Numbers in parentheses are percentages without considering the 93-base-deletion mutations. MF, mutation frequency. single-base-substitution mutations. One-third of the sponta- tion (+) or deletion (-) of bases were observed among those neous and two-thirds of the induced mutations are of this formed spontaneously. However, only deletion frameshifts type. Depurination enhances base-substitution mutagenesis (7/92) were observed among the induced mutations and, 31-fold above background to a frequency of 62.3 x 10-4. while these were enhanced 9.2-fold above spontaneous, the A second mutation that is increased in frequency is the 93- absolute frequency was small compared with base-substitu- base-deletion mutation. This event is presumably the result tion mutations (7.6% vs. 86%). No addition mutations were of recombination between the F' episome containing this ex- obtained, and the frequency of large deletion mutations (oth- act deletion (i.e., lacZAM15; see Fig. 1), and M13mp2. Fur- er than the 93-base deletion) was not increased. ther evidence that recombination is responsible is the obser- The spectrum of induced base substitution and frameshift vation that these deletion mutants have an associated ade- mutations is shown in Fig. 1. Of the seven frameshift muta- nine -* guanine base change at the EcoRI site (see Fig. 1) tions found, six represent the loss of a single base and one is representing the original base altered in the construction of a double-base loss. Four of the seven have lost a purine. The M13mp phage. Since this deletion results from the use of F' 79 single-base changes occur at 38 different positions and in lac-containing competent cells, these mutants need not be general reflect sequences known to be important for expres- considered further. Base substitutions thus represent an sion. even greater percentage of the total induced mutations The distribution of base-substitution mutants is not ran- (79/92, 86%). dom; 23% (18/79) are found in the 125-base operator/pro- Two other classes of mutations are increased in the in- moter region, while 77% (61/79) are found in the 129-base duced spectrum, double mutations and frameshifts. Three lacZ coding sequence. One "hotspot" is observed at the co- nontandem double mutations were observed, one of which dons for amino acids 16 and 17 of the lacZ gene. Nineteen (see Fig. 1, number 3, in the -35 promoter region) resembles mutants (24% of the total) were observed at the three guano- a similar mutation, at this same site, in the UV-induced spec- sines, representing only 4.4% of the currently known muta- trum (26). Considering the rarity of double mutations, the ble sites (ref. 26; this study) or just 1.2% of the 254-base existence of two similar nontandem double mutants resulting total. from two different mutagenic treatments suggests a common An analysis of the specificity of the 79 induced base-sub- mechanism of production inherent to the structure of the stitution mutations is presented in Table 4. Of 38 sites mutat- DNA (35), the specificity of the replication apparatus, or ed, 30 were positions of purines in the template strand, with both. Double mutations have also been detected twice a preference ofguanine over adenine sites mutated. Ofthe 79 among 32 depurination-induced amber revertants in OX174 base substitutions, 64 (81%) were at these 30 purine posi- DNA (21). They could reflect events at two different dam- tions. Transversions (72%) were 3-fold more frequent than aged sites in the same molecule or result from a targeted and transitions (28%), in contrast to the spontaneous base substi- an untargeted (semitargeted) event at some distance from the tutions, which were 79% transitions (data not shown). As- lesion. Frameshift mutations resulting from either the addi- suming that a misincorporation event occurred during the

A T T T TC A T T CT T T TCG T GCGCA ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTC TTTATGCTTC CGGCTCG7AT GlIjGTGTGGA ATTGTGAGCG GATAACAATT TCACACG AACAGCTATG * A3~SA3Ly A4 o) A 0 AT CT TT T A A A TT T T A T A C T TT T AT A AT T T C A C T T ATT T TT T A A T TT A TA TA T TT T TA ACC ATG ATT ACG AAT TCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC CCT GGC GTT ACC CAA CTT AAT CGC CTT GCA GCA CAT CCC CCT TTC GCC AGC TGG CGT AAT AGC GAA GAG GCC CGC A t t c CS A2 A2 A1l A A A +

FIG. 1. Induced mutational spectrum for base substitutions and frameshifts in M13mp2 lac DNA. The 5' -. 3' DNA sequence of the viral strand of the mutagenic target in M13mp2 is shown, from the first nucleotide after the laci termination codon through the coding sequence for amino acid 43 of the lacZ gene. Frameshifts are shown below the sequence, indicated by A. When a frameshift occurs in a run of two or more of the same base, the exact nucleotide lost is unknown and the A is centered under the run. Double mutations, linked by common subscripts, have the indicated change. For example, the first double mutation, indicated with the subscript 1, contains both a C-G -* AT and a G'C -+ CG transversion in the same molecule. Single-base-substitution mutations are shown above the wild-type sequence and indicate the nucleotide present in the viral strand. 0, CAP site; 0, -35 promoter; 0, -10 promoter; ®, transcription start; ®, operator site; 0, ribosome-binding site; (0, translation start; 0), position mutagenized to make the EcoRI site in the original mp2 construction; 0, first and last nucleotides of the 93-base deletion; @0, known mutable purine sites at which no base substitutions were observed in this study. Downloaded by guest on September 24, 2021 Genetics: Kunkel Proc. Natl. Acad. Sci. USA 81 (1984) 1497 Table 4. Specificity of induced base-substitution mutations insertion of adenine residues opposite apurinic sites (21, 32). The base-substitution specificity observed here for 38 sites is Sites Total Template Incorpo- similar; 59% of the mutants result from incorporation of Template mutated, mutations, mutation ration dAMP (Table 4). This specificity could result from several base no. no. Base No. event* factors. The possibility that the base immediately preceding Guanine 20 48 A 14 T or following ain AP site can be used as a template to deter- T 27 A mine the specificity of mutagenesis (by looping out the tem- C 7 G plate or daughter strand) can explain only 25% of the ob- Adenine 10 16 G 1 C served specificity. Furthermore, if looping out is a general T 13 A mechanism for bypass, one would expect to see a greater C 2 G increase in frameshift mutations than actually observed. Cytosine 6 13 T 7 A This mechanism thus seems unlikely. A 6 T Because an AP site is a noncoding lesion, incorporation G 0 C specificity could reflect nucleotide substrate concentrations Thymine 2 2 C 0 G in vivo. Treating cells with DNA-damaging agents results in A 2 T alterations in intracellular dNTP pools (37), the greatest ef- G 0 C fect being a 3- to 8-fold increase in the dATP pool. This in- *Assuming that misincorporation occurs during the first round of crease could be reflected in the specificity observed here. DNA replication in vivo. Alternatively, purified DNA polymerases are known to in- corporate dAMP opposite AP sites in vitro when equimolar dNTP concentrations are used (32, 38). Moreover, using sin- first round of replication in vivo, there is a preference for gle nucleoside triphosphates, Strauss et al. (33), found a sim- incorporation of adenine residues (47/79 or 59%). The re- ilar DNA polymerase incorporation specificity opposite maining incorporation specificity is in the order thymine apyrimidinic sites. Thus dAMP incorporation at AP sites (28%), guanine (11%), then cytosine (1%). may not simply be a consequence of pool imbalances but may reflect inherent properties of the AP site, the replication DISCUSSION apparatus, or both. The major goal of this work was to establish the spectrum of The transversions characteristic of adenine incorporation mutational events resulting from AP sites in DNA, to better opposite positions of template purines are thought to be the understand the available alternatives for processing a non- predominant mutagenic event induced by benzo[a]pyrene coding lesion. The data reported here establish both the fre- (39), and aflatoxin B1 (40), which produce primarily bulky quency and specificity of mutagenesis resulting from the in adducts in DNA. Such lesions could enhance mutagenesis viva SOS-dependent replication of unrepaired AP sites in through a common AP site intermediate, as suggested by the M13mp2 DNA. Mutagenesis results primarily from an in- data of Schaaper et al. (11) and Drinkwater et al. (9). Alter- crease in single-base-substitution errors. The absence of mu- natively, dATP may be the preferred substrate whenever tants arising from the addition of a large number of bases and base hydrogen bonding is not possible, regardless of the ac- the negligible effect on deletion mutations indicate that, at tual lesion. This implies that specificity is imposed by the least in this system, such events are not induced by AP sites. replication apparatus, a suggestion supported by the recent The spectrum of induced mutations yielded no addition observation that, in the absence of mutagen treatment, the frameshift mutants and only 8% deletion frameshifts. This primary base-substitution event in the lacI gene of E. coli low yield does not reflect a difference in target size for observed on constitutive expression of error-prone replica- frameshifts versus other events, since frameshift mutants are tion is a G-C T-A transversion (J. H. Miller and K. B. detectable at most sites in the lacZ coding sequence and at Low, personal communication). many sites in the operator/promoter sequences. Thus, fail- Adenine incorporation specificity has been suggested to ure to detect larger numbers of these events indicates that result from preferential binding of adenine residues to the they are not a major component of depurination-induced mu- DNA polymerase in the absence of template instruction (33). tagenesis. The fact that only deletion frameshifts were ob- Measurements with E. coli DNA polymerase I (ref. 41; bind- served suggests that looping out of the template strand con- ing preference, guanine > adenine > thymine > cytosine) taining the AP site may occur more frequently than looping and terminal deoxynucleotidyl transferase (ref. 42; binding out of the newly synthesized strand. preference, guanine = adenine > thymine > cytosine) do not The large absolute and relative increases in base-substitu- reflect the specificity observed in this study. However, simi- tion mutations (Table 3) provide a large number of mutants lar data are not available for other DNA polymerases, and for analysis of specificity under conditions in which most of other factors, such as exonucleolytic proofreading (32), are the mutants are induced. The small increase not dependent expected to modulate any potential correlation. This concept on SOS induction or damage to the DNA (Table 1) could deserves further study. result from rare bypass of AP sites under noninduced condi- Although incorporation of dAMP is the most frequent tions, untargeted mutagenesis, or lesions other than AP event, both dTMP and dGMP are incorporated at substantial sites. However, under the conditions used here to damage frequencies. Incorporation of dTMP occurred at potential the DNA, other lesions (17, 28, 29, 36) are expected to be apurinic sites in 14 mutants. However, this nucleotide is in- rare relative to AP sites. The results in Table 2 and the obser- corporated opposite positions of template pyrimidines eight vation that 79% of the sites mutated and 82% of the total times, resulting in transversions not seen even once in the single-base substitutions are at template purines are also spontaneous spectrum. These unique mutants could repre- consistent with AP sites being primarily responsible for the sent incorporation of dTMP opposite apyrimidinic sites, al- observed increase. This purine site specificity is not imposed though this would imply that apyrimidinic sites are produced by the DNA target; a recent study of UV mutagenesis using at frequencies somewhat greater than predicted from a previ- this same target gave opposite results; eight of nine sites mu- ous study (6). Alternatively, these mutants could result from tated and 32 of 35 single-base changes were at template py- untargeted events due to the specificity of error-prone repli- rimidines (26). cation. Previous studies on the reversion of three amber muta- Eleven transversions resulted from incorporation of tions in 4X174 DNA have indicated a strong preference for dGMP opposite potential apurinic sites. Two such transver- Downloaded by guest on September 24, 2021 1498 Genetics: Kunkel Proc. NatL. Acad. Sci. USA 81 (1984)

sions were observed among the depurination-induced rever- 4. Lindahl, T. & Andersson, A. (1972) Biochemistry 11, 3618- tants of the 4X174 amber 18 mutation (21). Although less 3623. frequent than mutants arising from adenine incorporation, 5. Grossman, L. & Grafstrom, R. (1982) Biochimie 64, 577-580. 6. T. Karlstrom, are rare not in at- Lindahl, & 0. (1973) Biochemistry 12, 5151- these mutants not and should be ignored 5154. tempting to understand what information is used to deter- 7. Margison, G. P., Capps, M. J., O'Connor, P. J. & Craig, mine the mutagenic outcome of a lesion. A. W. (1973) Chem. Biol. Interact. 6, 119-124. The distribution of mutants is not random. As in measure- 8. Lawley, P. D. & Brookes, P. (1963) Biochem. J. 89, 127-138. ments of depurination-induced reversion of kX174 amber 9. Drinkwater, N. R., Miller, E. C. & Miller, J. A. (1980) Bio- mutants (21, 32), site-specific differences are apparent here. chemistry 19, 5087-5092. For example, the middle guanine residue in the hotspot (Fig. 10. Strauss, B., Scudiero, D. & Henderson, E. (1975) in Molecular 1) gave nine mutants. In contrast, no mutants were observed Mechanisms for Repair ofDNA, eds. Hanawalt, P. C. & Set- at the adenine residue four nucleotides away (5' direction, low, R. B. (Plenum, New York), Part A, pp. 13-24. Fig. 1), which is known (J. E, LeClerc and N. L. 11. Schaaper, R. M., Glickman, B. W. & Loeb, L. A. (1982) Can- Istock, cer Res. 42, 3480-3485. personal communication) to be mutable by the most frequent 12. Lindahl, T. (1982) Annu. Rev. Biochem. 51, 61-87. incorporation event associated with depurination (incorpo- 13. Duker, N. J., Hart, D. M. & Grant, C. L. (1982) Mutat. Res. ration of adenine). Based on current information, five purine 103, 101-106. sites (Fig. 1) known to produce a mutant phenotype are not 14. Deutsch, W. A. & Linn, S. (1979) Proc. Natl. Acad. Sci. USA found in this spectrum, three of which could have resulted 76, 141-144. from the most frequently expected transversion. This appar- 15. Brookes, P. & Lawley, P. D. (1963) Biochem. J. 89, 138-144. ent nonrandom distribution of mutants may reflect the rela- 16. Lawley, P. D. & Martin, N. C. (1975) Biochem. J. 145, 85-91. tively small number of mutants analyzed or could reflect 17. Drake, J. W. & Baltz, R. H. (1976) Annu. Rev. Biochem. 45, 11-37. more important factors. Of eight purine sites yielding three 18. Schaaper, R. M. & Loeb, L. A. (1981) Proc. Natl. Acad. Sci. or more mutations, seven have purines immediately adja- USA 78, 1773-1777. cent, and the hotspot is in a run of seven purines. Perhaps 19. Shearman, C. W. & Loeb, L. A. (1979) J. Mol. Biol. 128, 197- depurination may occur more readily in a purine-rich se- 218. quence. Differences in the rate of depurination of double- 20. Kunkel, T. A., Shearman, C. W. & Loeb, L. A. (1981) Nature and single-stranded DNA are known (3) and guanine resi- (London) 291, 349-351. dues depurinate more readily than adenine residues (3). This 21. Schaaper, R. M., Kunkel, T. A. & Loeb, L. A. (1983) Proc. may explain the preference for guanine versus adenine sites Natl. Acad. Sci. USA 80, 487-491. mutated (Table 4). Alternatively the nonrandom distribution 22. Messing, J., Gronenborn, B., Muller-Hill, B. & Hofschneider, of mutants may reflect differences in the frequency of by- P. H. (1977) Proc. Natl. Acad. Sci. USA 74, 3542-3546. 23. Kane, C. M. pass of apurinic sites (21). & Linn, S. (1981) J. Biol. Chem. 256, 3405-3414. 24. Taketo, A. (1972) J. Biol. Chem. 72, 973-979. AP sites are highly mutagenic under SOS-induced condi- 25. Langley, K. E., Villarejo, M. R., Fowler, A. V., Zamenhof, tions, resulting in 0.2% mutants per lethal hit (Table 3) for a P. J. & Zabin, I. (1975) Proc. Natl. Acad. Sci. USA 72, 1254- target DNA sequence of only 250 bases. This is 5- to 10-fold 1257. greater per lethal hit than the SOS-dependent mutagenesis of 26. LeClerc, J. E. & Istock, N. L. (1982) Nature (London) 297, this same target resulting from UV irradiation of intact 596-598. M13mp2 phage (26). This highly mutagenic response pre- 27. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. sumably results from two properties of AP sites. Unlike Acad. Sci. USA 74, 5463-5467. modified bases, which may retain some correct base-coding 28. Karran, P. & Lindahl, T. (1978) J. Biol. Chem. 253, 5877-5879. 29. Lindahl, T. & Nyberg, J. B. (1974) Biochemistry 13, 3405- potential, AP sites are truly (base) noncoding lesions. In ad- 3410. dition, AP sites probably distort DNA to a lesser extent and 30. Little, J. & Mount, D. (1982) Cell 29, 11-22. sterically constrain polymerization less than certain bulky 31. Schaaper, R. M., Glickman, B. W. & Loeb, L. A. (1982) Mu- adducts do (33, 43, 44), resulting in more frequent bypass. tat. Res. 106, 1-9. The estimates of AP site bypass frequency in vitro are in fact 32. Kunkel, T. A., Schaaper, R. M. & Loeb, L. A. (1983) Bio- quite high for several DNA polymerases (20, 21, 32, 38), in chemistry 22, 2378-2384. contrast to the results obtained with bulky base modifica- 33. Strauss, B., Rabkin, S., Sagher, D. & Moore, P. (1982) Biochi- tions (33, 45). These properties of AP sites, in combination mie 64, 829-838. with the potentially high frequency of their production, make 34. Lockhart, M. L., Deutch, J. F., Yamaura, I., Cavalieri, L. F. these lesions interesting for future studies of repair. & Rosenberg, B. H. (1982) Chem. Biol. Interact. 42, 85-95. 35. Ripley, L. S. (1982) Proc. Natl. Acad. Sci. USA 79, 4128- 4132. 36. Bingham, P. M., Baltz, R. H., Ripley, L. S. & Drake, J. W. I would like to express appreciation to J. Motto and J. Liu for (1976) Proc. Natl. Acad. Sci. USA 73, 1273-1277. assistance in DNA sequence analysis and to M. Volkert, R. 37. Das, S. & Loeb, L. A. (1984) Mutat. Res., in press. Schaaper, B. Glickman, and J. Drake of this Institute for critical 38. Boiteux, S. & Laval, J. (1982) Biochemistry 21, 6746-6751. evaluation of the manuscript. The gift of strains and the advice of 39. Eisenstadt, E., Warren, A. J., Porter, J., Atkins, D. & Miller, Eugene LeClerc (University of Rochester) during the initial stages J. H. (1982) Proc. Natl. Acad. Sci. USA 79, 1945-1949. of this work were most helpful and are gratefully acknowledged. 40. Foster, P. L., Eisenstadt, E. & Miller, J. H. (1983) Proc. Natl. Acad. Sci. USA 80, 2695-2698. 41. Englund, P. T., Huberman, J. A., Jovin, T. M. & Kornberg, 1. Lindahl, T. (1979) Prog. Nucleic Acid Res. Mol. Biol. 22, 135- A. (1969) J. Biol. Chem. 244, 3038-3044. 192. 42. Kato, K., Goncalves, J., Houts, G. & Bollum, F. (1967) J. 2. Kunkel, T. A., Schaaper, R. M., James, E. & Loeb, L. A. Biol. Chem. 242, 2780-2789. (1981) in Induced Mutagenesis: Molecular Mechanisms and 43. Grunberger, D., Blobstein, S. & Weinstein, I. (1974) J. Mol. Their Implications for Environmental Protection, eds. Law- Biol. 83, 459-468. rence, C. W., Prakash, L, & Sherman, F. (Plenum, New 44. Fuchs, R. P. P. (1975) Nature (London) 257, 151-152. York), Vol. 23, pp. 63-82. 45. Moore, P. D., Rabkin, S. D. & Strauss, B. S. (1980) Nucleic 3. Lindahl, T. & Nyberg, B. (1972) Biochemistry 11, 3610-3618. Acids Res. 8, 4473-4484. Downloaded by guest on September 24, 2021