Proc. Natl. Acad. Sci. USAVol. 92, pp. 2234-2238, March 1995Genetics

DNA nick processing by exonuclease and polymerase activities ofbacteriophage T4 DNA polymerase accounts for acridine-inducedmutation specificities in T4

(frameshift/mutagenesis/9-aminoacridine/T4 topoisomerase II)

VICKI L. KAISER AND LYNN S. RIPLEY*Department of Microbiology and Molecular Genetics, New Jersey Medical School and Graduate School of Biomedical Sciences, University of Medicine andDentistry of New Jersey, Newark, NJ 07103

Communicated by Allan M. Campbell, Stanford University, Stanford, CA, August 29, 1994 (received for review May 24, 1994)

ABSTRACT Acridine-induced frameshift mutagenesis inbacteriophage T4 has been shown to be dependent on T4topoisomerase. In the absence of a functional T4 topoisomer-ase, in vivo acridine-induced mutagenesis is reduced to back-ground levels. Further, the in vivo sites of acridine-induceddeletions and duplications correlate precisely with in vitrosites of acridine-induced T4 topoisomerase cleavage. Thesecorrelations suggest that acridine-induced discontinuities in-troduced by topoisomerase could be processed into frameshiftmutations. The induced mutations at these sites have aspecific arrangement about the cleavage site. Deletions occuradjacent to the 3' end and duplications occur adjacent to the5' end of the cleaved bond. It was proposed that at the nick,deletions could be produced by the 3' -* 5' removal of basesby DNA polymerase-associated exonuclease and duplicationscould be produced by the 5' ->3' templated addition of bases.We have tested in vivo for T4 DNA polymerase involvement innick processing, using T4 phage having DNA polymeraseswith altered ratios of exonuclease to polymerase activities. Wepredicted that the ratios of the deletion to duplication muta-tions induced by acridines in these polymerase mutant strainswould reflect the altered exonuclease/polymerase ratios ofthe mutant T4 DNA polymerases. The results support thisprediction, confirming that the two activities of the T4 DNApolymerase contribute to mutagenesis. The experiments showthat the influence of T4 DNA polymerase in acridine-inducedmutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplica-tions by a mechanism that does not involve DNA slippage.

Initial studies of protein sequence changes produced by spon-taneous and acridine-induced frameshift mutations in thelysozyme gene of bacteriophage T4 revealed that single basedeletions or duplications frequently occurred within A-T runs(1). It was therefore proposed that both deletions and dupli-cations occur because one DNA strand could stably misalignupon the complementary strand within the base run in twoways: a bulged template strand produces a deletion in theelongating DNA, whereas a bulged elongated strand producesa duplication (1). Acridine-induced mutations generally con-sistent with the predictions of this slippage model were ob-served in the Escherichia coli lacI gene (2, 3) and in bacterio-phage A (4). In these systems, the induced frameshifts occurpreferentially in G-C runs.

Subsequent DNA sequencing of acridine-induced mutantsin the T4 lysozyme gene identified frequent frameshifts thatwere neither within nor adjacent to base runs or other repeatsand therefore could not be explained by DNA strand slippage(5, 6). The slippage model also fails to explain the specificities

of many acridine-induced frameshift mutations that arise athotspot sites in the rIIB and thymidylate synthase genes of T4(7, 8). For example, a frequent mutation within the rIIBsequence 5'-AAATTGTTAAACT-3' is the duplication of theG. Because this G is flanked on both sides by two T residues,the G cannot slip to form a complementary base pair. More-over, despite the high frequency ofG duplications, G deletionsare not induced as would be predicted by slippage.The specificities of deletions and duplications at the rIIB

hotspot site are consistent with a different model. In this model,bases are deleted or duplicated by exonuclease or by DNApolymerase, respectively, at the 3' ends of acridine-induced, T4topoisomerase-generated nicks (Fig. 1) (9). The frameshift mu-tations are created by religation of the shortened or extended 3'end to the original 5' end of the nick (10). In vitro, the productsof acridine-induced, topoisomerase-generated nicks are a free3'-hydroxyl end and a 5'-phosphate end that remains covalentlylinked to the topoisomerase (11). The sites at which this in vitroreaction occur in T4 DNA correlate with the preferred sites of invivo mutations (7, 10, 12).

In contrast to the slippage model, this model accountsentirely for the specificity of mutations arising at the T4 rIIBhotspot (7, 10). For example, this nick-processing model,coupled with the observed sites of acridine-induced cleavage,predicts G duplications and accounts for the absence of Gdeletions discussed above. Alternative processing at the samenick predicts deletion, but not the duplication, of the T 5' tothe G. Consistent with this prediction, deletions, but notduplications, are frequently induced. Moreover, the prepon-derance of deletions is inconsistent with the predictions ofslippage, which predicts duplications. The surrounding DNAsequence provides further evidence that A-run sites are notautomatic hotspots for acridine-induced frameshifts. The A-run upstream of the G is not cleaved in vitro and mutations arenot increased by acridines; the A run downstream of the G iscleaved and mutations are increased by acridines (10).

In support of the proposed role of the T4 topoisomerase inacridine mutagenesis: (i) mutagenesis is abolished in acridine-treated T4 phage having an amber mutation in one subunit of thetopoisomerase (10); (ii) the in vitro acridine-induced topoisomer-ase cleavage sites correspond precisely to the positions of in vivoacridine-induced frameshift sites (7, 10); and (iii) when newtopoisomerase cleavage sites are created by introducing DNAsequence changes in the vicinity of the rIIB hotspot, acridine-induced frameshifts arise at these new sites with exactly thespecificities predicted by the nick-processing model (12).

Because T4 DNA polymerase contains both a 3' exonucle-ase and polymerase activities, this enzyme alone might account

Abbreviation: 9-AA, 9-aminoacridine.*To whom reprint requests should be addressed at: Department ofMicrobiology and Molecular Genetics, University of Medicine andDentistry of New Jersey, 185 South Orange Avenue, Newark, NJ07103.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. NatL Acad Sci USA 92 (1995) 2235

G~~TTA

5. 7 (M A A A 7 7TOM A A a T a3 T3

OATYTAACAATTTQACA

ts J +GT_3 > Et, *oonuom.as polymerization

FIG. 1. Model for acridine-induced deletions and duplications inT4. Topoisomerase (Topo) has nicked the DNA and remains co-

valently linked through the phosphate to the 5' end of the DNA. Thefree 3'-hydroxyl end serves, as a substrate for the T4 DNA polymerase.One (e.g., -T) or more bases can be removed from the 3' end by actionof the 3' -- 5' exonuclease. Religation of this processed end with the

topoisomerase-linked 5' end would result in a deletion. Alternatively,by using the bottom strand as a template, one or more (e.g., +GT)bases can be added to the 3' end by action of the polymerase.Religation of this processed end to the topoisomerase-linked 5' endwould result in a duplication.

for both the 3' -* 5' deletion and 5' -- 3' duplicationspecificities exhibited by the acridine-induced rIIB mutationswith respect to the topoisomerase-mediated nicks. We havetested for this possibility by using T4 DNA polymerase mutantswith altered ratios of exonuclease to polymerase activities. T4DNA polymerase involvement predicts that the in vivo ratio ofdeletion to duplication mutations should reflect the alteredratios of the competing exonuclease and polymerase activitiesof the mutant DNA polymerases if the T4 DNA polymerase isinvolved in the production of both deletions and duplications.The results confirm the active role of the T4 DNA polymerasein acridine-induced mutagenesis by demonstrating the pre-dicted influence of diverse polymerase alleles on acridine-induced mutational specificity.

MATERIALS AND METHODSBacteriophage and E. coli Strains. The T4 DNA polymerase

mutants tsL141, tsL56, and tsL98 were originally isolated byEpstein et al. (13) in bacteriophage T4D and were backcrossedfour to five times to T4B (14). The phage strain T4B was usedas a wild-type DNA polymerase control. The T4 rIIB muta-tions FC11 (-1 frameshift,- -A 458-462) and FC47 (+1frameshift, -TT, +CTG 561-562) (numbering as in ref. 7)were separately crossed into each polymerase background as

described (14). The use of the rIIB gene for detection offrameshifts has been described (14). E. coli BB, which ispermissive for rIIB mutants, was used as a host during allmutagenesis (7). E. coli K38-A, which contains a A prophageand is nonpermissive for rIIB mutants, was used to detectframeshift revertants of FC11 and FC47 (7).

All phage plating was done on 35 ml of Drake agar (5 g ofNaCl, 0.2 g of dextrose, 1 g of yeast extract, 10 g of Bacto-tryptone, 10 g of Bacto-agar and water to 1 liter) with 2.5 mlsoft agar overlay (17 g of Drake agar and water to 1 liter). Allphage were treated and grown at 30°C in M9/CA broth [3 gof KH2PO4, 6 g of Na2HPO4, 1 g of NH4Cl, 3.5 g of NaCl, and0.16 mg of FeCl3 in water to 1 liter, to which separatelyautoclaved solutions of (i) glucose (4 mg/ml) and MgSO4 (266gg/ml) and (ii) Casamino acids (8 mg/ml) were added].

9-Aminoacridine (9-AA) Mutagenesis. 9-AA (Aldrich) was

stored as a stock solution (1 mg/ml in sterile water) in a

foil-wrapped bottle at 20°C. Mutagenesis was performed onthree independent stocks isolated from each polymerase back-ground to control for possible mutational "jackpots" arisingwithin a single stock. Parallel and identical treatments were

performed on all three stocks for each polymerase back-ground. One wild-type polymerase phage stock was treated

with each set of mutant polymerase phage stocks to control forday-to-day variability.The T4 phage stocks used in mutagenesis were diluted with

fresh M9/CA to 2.5 x 1010 phage per ml. E. coli BB cells weregrown to midlogarithmic phase (2 x 108 cells per ml), con-centrated to 5 x 108 cells per ml by centrifugation at 4°C in aSorvall SS-34 rotor at 5000 rpm for 10 min, and then resus-pended in fresh cold M9/CA. Infection was initiated by addingconcentrated BB cells to each T4 phage stock at a multiplicityof infection of 5 and incubating them on a rotary shaker at30°C. Two infection tubes, each individually timed, wereinitiated for each phage stock. After 13 min on a rotary watershaker, one infection mixture was diluted 1:10 into prewarmedM9/CA (spontaneous mutation control) and the other wasdiluted 1:10 into prewarmed M9/CA containing 9-AA (32,ug/ml). After shaking for 30 min, the control tubes werediluted 1:10 into prewarmed M9/CA and the 9-AA treatmenttubes were diluted to a final 9-AA concentration of 0.2 ,ug/mlinto prewarmed M9/CA. This dilution was made to minimize9-AA interference with phage development (15). After incu-bation for an additional 40 min, lysis was completed by theaddition of chloroform.The revertant (frameshift mutant) frequencies were deter-

mined using differential plating on E. coli strains. The numberof revertants was obtained by plating on K38 cells and the totalnumber of phage was obtained by plating on E. coli BB cells.The revertant frequencies were calculated as the ratio of theK38 titer to the BB titer. Revertants were picked from the K38plates and purified by streaking on BB cells. A single purifiedplaque was grown to high titer for sequence analysis. A 9-AAdose-response curve was made for each DNA polymeraseallele. These were performed as described above with thefollowing modifications. For each mutant DNA polymeraseallele, one wild-type polymerase control and one mutant DNApolymerase phage stock were run in parallel. One infectiontube was initiated for each concentration of 9-AA tested (0, 2,4, 8, 16, 32, 64, and 100 ,ug/ml) and, after 9-AA incubation,each treatment tube was diluted to a final 9-AA concentrationof 0.2 ,ug/ml.DNA Sequencing. The DNA sequences of most of the T4

revertants were determined by 32P-end-labeled oligonucleo-tide probing (10). Exactly matched oligonucleotides weredetected by autoradiography after washing the filter at apredetermined temperature (10, 12). The oligonucleotideprobes used were complementary to either wild-type or pre-viously characterized mutant sequences (7, 10). All the mutantsequences described in this paper fail to hybridize to a probecomplementary to wild-type T4 rIIB sequence positions 473-487. The sequences of mutants not determined by probingwere determined by direct sequencing of T4 genomic DNA,using dideoxy sequencing (16) and PCR amplification (M.Masurekar, V.L.K., and L.S.R., unpublished work).

RESULTS AND DISCUSSION9-AA induces frameshifts in T4 (17) and interferes with phagematuration and DNA processing in vivo (15, 18). Additional invitro studies suggest that 9-AA interferes with the activities ofT4 DNA polymerase by mechanisms that may reflect directinteraction with the polymerase protein (19). In view of therange and potential polymerase-specific effects of 9-AA treat-ments, the effect of 9-AA dose on phage burst size andmutagenesis for each DNA polymerase allele was measured.9-AA was mutagenic and produced similarly decreased aver-age burst sizes with increased concentration in all polymerasebackgrounds, confirming that there were no exceptional poly-merase-specific drug effects (data not shown). 9-AA at 32,ug/ml produced a high amount of mutagenesis with a modestdecrease in phage burst size and was therefore used insubsequent mutagenesis experiments.

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Six independent stocks were mutagenized with 9-AA foreach T4 DNA polymerase allele examined. Three of the stockshad an initial -1 (FCJJ) frameshift providing an assay forgenetic + 1 frameshifts. Three of the stocks had an initial + 1(FC47) frameshift providing an assay for genetic -1 frame-shifts. Fig. 2 shows a map of the rIIB spontaneous and9-AA-induced mutants and the average revertant frequenciesfor each DNA polymerase allele tested. 9-AA treatmentsignificantly increased the average revertant frequencies ofeach T4 DNA polymerase allele over the untreated controls.Consistent with previous observations, the mutant DNA poly-merase alleles produce elevated spontaneous revertant fre-quencies compared with the wild-type DNA polymerase (14).The characterization of polymerase effects on frameshift

specificity was based on the sequences of 1800 revertantsinduced by 9-AA in four DNA polymerase backgrounds.Earlier studies have shown that the acridines 4'-(9-acridinylamino)methanesulfon-m-anisidide and proflavin pro-duce high frequencies of frameshifts in the DNA sequencebetween 473 and 487 in a wild-type polymerase background.This study shows that 9-AA also produces high frequencies offrameshifts in this same region in all DNA polymerase back-grounds tested. This region is not a hotspot for spontaneous

L141WTL98L56

Revertarfrequencl(x105)

37,34

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16,16

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L141 0.5, 1.8WT 0.16 OA

L98 0.2, 1

y

9.AA induced mutants

0,0 240,156 2,1 0,4 0,3

10,1 231,114 1,0 0,0 1,0

24,37 214, 195 0,ND 0,4 ND, 1

50,73 145,145 1,ND 1,3 0,0

* .

I i i 4 i i I i-Ii i

0,11 9,3 22,2 0,15 54,70

615 21,5 0,0 7,6 5,20

115,192 3,7 0,ND 4,12 ND, 13

L56 0.7,3.0 134,159 42,0 1,ND 27, 30 4,17

1,0

0,1

ND, 12

13,7

Oiler

10,8

18,7

23,16

35,33

i- -- I1

7,76,1

NR 34

172,68161,93

141,3

19,31 35,27

Spontaneous mutants

FIG. 2. Map of 9-AA-induced and spontaneous frameshifts se-lected as revertants of FCII and FC47 in the rIIB gene of bacterio-phage T4. The solid line and its numerical coordinates represent therIIB DNA sequence in which frameshifts were detected. Open barsshow the positions of the 15-mer oligonucleotides used to map theposition of the mutants. 9-AA-induced and spontaneous mutants areshown above and below the sequence line, respectively. The pairs ofnumbers associated with each oligonucleotide position indicate thenumber of frameshifts that suppress FCI (a -1 frameshift) or FC47(a + 1 frameshift). Revertants of FCI I are genetically + 1 (e.g., + 1 or

+4 duplications or -2 or -5 deletions). Revertants of FC47 are

genetically -1 (e.g., +2 or +5 duplications or -1 or -4 deletions). Thenumber of revertants analyzed for either FCII or FC47 is calculatedby summing the numbers associated with each oligonucleotide with the"other" category. Approximately 85 revertants were chosen fromthree independent stocks for each genotype; only two stocks werechosen for L141/FC47 and only 40 revertants were selected from eachwild-type (WT) FC47 stock. The average revertant frequencies weremeasured in the same stocks from which frameshifts were selected forsequence analysis. One line of entries is shown for each polymerasegenotype tested. ND indicates that specific probings were not done.Positions of the mutants shown in the "other" category have not beendetermined and are predicted to lie in unprobed regions of the mapor be undetectable by the oligonucleotides. Star indicates the positionof the oligonucleotide complementary to the wild-type sequencebetween 473 and 485 that includes the 9-AA-induced frameshifthotspot sequence (see Fig. 1). A G-C run of 3 bp is located at 507-509and is a "warm spot" for spontaneous 1-bp deletions and duplications(20). 9-AA does not stimulate frameshifts in this run in any polymerasebackground.

frameshifts in any polymerase background; therefore sponta-neous frameshifts do not interfere with analysis of 9-AA-induced frameshift specificity in the vicinity of the hotspot (seebelow).The mutant T4 DNA polymerase alleles used in this study

were selected because previous characterization of the purifiedproteins demonstrated their altered ratios of exonuclease topolymerase activity in vitro (21). Subsequent studies (22-25)have addressed some of the biochemical mechanisms thatunderlie these altered ratios. As measured in nucleotide turn-over assays, mutant T4 DNA polymerase L141 has an in-creased ratio of exonuclease to polymerase activity, while L98and L56 have decreased ratios of exonuclease to polymeraseactivities. Because of the proposed roles of exonuclease andpolymerase activities in deletion and duplication mutagenesis,respectively, a demonstration that the L141 allele increasesdeletion/duplication ratios while the L98 and L56 allelesdecrease these ratios would implicate both T4 DNA poly-merase activities in nick processing during 9-AA-inducedmutagenesis.

Eight tests of the relationship between exonuclease andpolymerase activities and frameshift specificity were made.These provide a total of six comparisons of the 9-AA-induceddeletion/duplication ratios of the three mutant T4 DNApolymerase alleles with the same ratios of T4 wild-type DNApolymerase at two topoisomerase nick sites. These deletion/duplication ratios are summarized in Fig. 3 along with thedetailed analysis of acridine-induced frameshift specificity andfrequency. Fig. 4 shows the sample-to-sample deviation of thedeletion/duplication ratios shown in Fig. 3 and the totalnumber of deletion and duplication mutations used to calcu-late each ratio.L141 increased the deletion/duplication ratios at both nick

sites when compared with the wild-type T4 DNA polymerase,as predicted by its high relative exonuclease activity (Fig. 3,compare columns 1 and 2 for both strands). In contrast, L98decreased the deletion/duplication ratios at the top-strandnick (compare column 3 with column 2 for the top strand) andL56 decreased the deletion/duplication ratio at the bottom-strand nick (compare column 4 with column 2 for the bottomstrand) as predicted by their lower relative exonuclease activ-ities. Thus, four of the six comparisons produced the exactspecificity changes predicted if T4 DNA polymerase and itsexonuclease convert DNA nicks into frameshift mutations.There was no detectable influence on the deletion/duplicationratios of the L98 allele at the bottom-strand site and the L56allele at the top-strand site (compare column 3 for the bottomstrand and column 4 for the top strand with column 2,respectively). These last two comparisons still do not precludethe T4 DNA polymerase and exonuclease activities in nickprocessing. For example, neither mutant polymerase produceda change in a direction opposite to that predicted, neither nicksite was routinely an exceptional site, and the exceptional sitesfor the L98 the L56 polymerases were different. Instead, thesetwo exceptional comparisons suggest that the relative T4 DNApolymerase activities at DNA nicks by the L98 or L56 allelesmay not differ substantially from the wild-type DNA poly-merase allele and are sensitive to DNA sequence context invivo. This interpretation is supported by the observation thatthe wild-type DNA polymerase itself clearly exhibits differentdeletion/duplication ratios at the two nick sites examined(Figs. 3 and 4).The mutant DNA polymerase alleles usually influence the

frequency of both deletions and duplications compared to thewild-type allele (Fig. 3). There are two exceptions. The L141and L56 alleles change only the deletion frequencies at thebottom-strand site. Notably, the deletions increase and de-crease, respectively, as predicted from the relative exonucleaseactivities expected for the L141 and L56 polymerases.

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FIG. 3. Summary of strand-specific 9-AA-induced deletion/duplication ratios for each DNA polymerase allele and the spectrumof 9-AA-induced frameshift sequences adjacent to the topoisomerasenick sites at the rIIB hotspot and their frequencies. The four poly-merase alleles used in the experiments head each column (WT, wild-type). Arrows beneath the allele names represent the exonuclease andpolymerase; their sizes (not to scale) indicate the direction of in vitroactivity changes in the mutants. The numbered DNA sequence (centerright) shows the DNA context surrounding the topoisomerase cleavagesites studied. The downward arrow (between T and G) represents thenick site in the top (nontranscribed) strand and the upward arrow(between T and T) represents the position of the nick in the bottom(transcribed) strand. Induced frameshifts have bases deleted or du-plicated beginning at these nick sites. The specific bases deleted or

duplicated at the top strand nick are shown above the startingsequence; bases complementary to those deleted or duplicated at thebottom strand nick are shown below this sequence. Sequence changeswhich suppress FCII are in bold type, while sequence changes thatsuppress FC47 are in lighter type. The induced frequency (x 108) ofeach frameshift sequence is given for each DNA polymerase allele;dashes indicate cases where no example of the genotype was detectedin the mutants collected. The figures are corrected for small ( 13)contributions from the spontaneous mutants. In one L98 stock a smalljackpot (7 of 86 revertants selected) in a single stock led to thesubtraction of a frequency of 50 for the -A (482-484) mutants. Thenumber of mutants sequenced for each mutation can be calculated bydividing the induced frequency reported in this table by the averagerevertant frequency for that genotype (from Fig. 2) and multiplyingthat fraction by the total number of revertants analyzed for thatgenotype (from Fig. 2). The deletion/duplication ratios for eachpolymerase allele at each nick site are shown below each column offrequencies for the top and bottom strands. Each ratio was calculatedby separately summing the frequencies of deletion and duplicationframeshifts adjacent to each nick site. The -1, + 1, and +2 frameshiftswere identified by oligonucleotide probing; the remainder were se-quenced. The +GTTA mutation is adjacent to both topoisomerasenick sites and therefore, could have arisen by polymerization at eithernick site. This frameshift is attributed to the top-strand nick becauselonger mutations occurred at this site in all polymerase backgrounds.However, there is minimal effect on the calculated ratios shown evenin the extreme case when the entire frequency of +GTTA is attributedto the bottom-strand nick site.

The deletion/duplication ratios of mutant alleles are distin-guishable from those produced by the wild-type polymeraseexcept for the ratios at the L98 bottom-strand site and the L56top-strand site (Fig. 4). This is not due to the failure of thesemutant DNA polymerase alleles to influence frameshift fre-quencies (Fig. 3). Both alleles influence both deletion andduplication frequencies despite the absence of a detectablechange in the deletion/duplication ratio. This result supportsthe interpretation that mutagenic nick processing is largelymediated by the T4 DNA polymerase, even though the ratio of

(0o 1.4

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1: 2.5

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(17, 89) _ 1: 7f i "|T (17, 67)

1:14 - 1:14(13, 225) 1: 37 (14 179

-'76 (6, 239) '

T B T B T B T B

L141 WT L98 L56' T4 DNA polymerase allele

FIG. 4. Mean and variance of strand-specific 9-AA induced dele-tion/duplication ratios for each DNA polymerase allele. Normalizedratios from Fig. 3 for top-strand nick sites (o) and bottom-strand ratios(-) are shown above T and B on the x axis. Bars show the calculatedstandard deviation for each ratio. The two numbers in parenthesesabove each bar are the number of independent sequenced deletion andduplication mutants, respectively, used to calculate the ratio. Twomajor sources of experimental variation are expected. One source isday-to-day variation in the calculated revertant frequency, itself a ratioof two experimentally determined numbers as described in Materialsand Methods. This variation was minimized by using the average, ratherthan stock-specific, revertant frequencies to calculate the revertantfrequencies of deletions and duplications. Deletion and duplicationfrequencies were separately calculated for FCI and FC47 stocks andwere then combined to produce the final site-specific deletion/duplication ratios for each polymerase allele. This approach minimizesthe impact of stock-specific coincidence of a high or low number offrameshifts having a specific deletion or duplication sequence with astock having an unusually high or low total revertant frequency. Asecond source of variation is the sample-to-sample variation in thedistribution of deletions and duplications. This variation was mini-mized by sequencing large numbers of mutants collected from mul-tiple, independent experiments. Sample-to-sample variance is pre-sented here as the standard deviation of the final mean. The calcu-lation of the standard deviation was made by comparing the deletion/duplication ratio calculated from all mutant frequencies for apolymerase allele (Fig. 3) with the set of ratios calculated fromfrequencies generated by pairing each 9-AA-treated stock of FCI1with each stock of FC47 for that polymerase allele. The pairwisecomparisons individually use a fraction of the data and create a set ofeither six or nine ratios for each mean. The estimated variance is ameasure of the internal consistency of the independently collectedportions of the data. As described in the text, four of the six final ratioscalculated in mutant polymerase backgrounds clearly differ from theratio calculated in the wild-type (WT) polymerase background. Re-calculation of the deviations using stock-specific revertant frequencies(data not shown) produced only slightly increased estimates of vari-ation in the final deletion/duplication ratios, suggesting that stock-specific revertant frequencies were not an important source of vari-ation in these measurements.

competing activities for a mutant allele may not differ sub-stantially from that of the wild-type polymerase at some sites.This observation, coupled with the distinct differences be-tween top- and bottom-strand ratios for the wild-type DNApolymerase, illustrates the strong influence of DNA sequenceon the molecular details that can influence mutagenic speci-ficity as well as frequency.The 1422 frameshift mutants described in Fig. 4 lie between

positions 473 and 487 and have precisely the DNA sequencespredicted by the proposed nick-processing model at the in vitronick sites. Fig. 5 shows eight different sequences of 20 (1.4%)exceptional frameshifts that were detected. Above the DNAsequence are 7 frameshifts which lie adjacent to the acridine-induced topoisomerase sites but have sequences that are notpredicted by templated elongation of the 3' end.The remaining exceptional frameshifts (Fig. 5) are not

adjacent to the nick sites detected in in vitro cleavage exper-

L141 WT L98 L56 9-AA-induced Mutations_ - 3-50 AAGCTGAAATT--- 100 - AAATT-

200 - - - AATT-1,500 400 100 200 TT-6,600 2,800 200 1,000 T-

13,800 16,900 5,000 9,800 tG6,600 25,500 9,00010,000 +GT400 700 500 1,400 +GTTA- 300 - 100 +GTTAA300 1,700 250 900 +GTTAAACTop Strand Ratios

1 :2.51 :14 3711 :14

466 486

A C A A G C T G A A A T T - G T T A - A A C TTG TTC G A CTTTAA- CAAT-TTGA

400 600 400 200 TA+16,200 12,600 7,000 11,400 A+

15,500 3,100 1,900 1,300 -A1,100 900 400 200 -AA1,300O - 200 200 -AACTBottom Strand Ratios

1:0.91 1:3 1 :3 1 :7.

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L141 WT L98 L56 Exceptional mutationsKoi>< 4E>_ I> O > adjacent to nick sites

1 0 0 0 T-/ + GTo O 1 1 T +O O 0 2 + Co o 0 1 + AO O 0 C+_

473 t 487

GAAATT-GTTA-AACTG

O O 0 3 -GO O 1 3 +AO O 1 3 tCO O 2 0 + CT

Exceptional mutations notadjacent to nick sites

FIG. 5. Genotypes of exceptional frameshift mutations producedin the vicinity of the rIIB hotspot site after treatment with 9-AA.Arrows under the polymerase alleles illustrate relative exonucleaseand polymerase activities. The wild-type (WT) rIIB sequence between473 and 487 is shown. Arrows above and below the sequence representthe topoisomerase nick sites for the top and bottom DNA strands,respectively. The number of mutants having each frameshift sequenceis indicated. Mismatched nucleotide incorporation before religationexplains the +T, +C, and +A insertions between 478 and 479 and the+C insertion between 482 and 483. Slippage could alternativelyexplain the +T insertions. The -T, +GT genotype may well be theconsequence of two mutations: +GT is an expected induced mutationin a -T strain, and -T is an expected mutation in a +GT strain (12).

iments (10). These frameshifts may have been induced at sitesnicked infrequently by topoisomerase or may be due tobackground mutagenesis. For example, studies of weak cleav-age sites induced by 4'-(9-acridinylamino)methanesulfon-m-anisidide correlated with weak mutagenesis sites (12); in vitrocleavage with 9-AA has not been measured. A clear contri-bution of background mutagenesis are the three +A frame-shifts (474-476) in the L56 polymerase background. All threeof these mutants were found in one mutagenized stock, andthis stock was found to be a jackpot for spontaneous +Aframeshifts at this site (41 +A sequences among 87 sponta-neous frameshifts sequenced). None of the remaining excep-tional sequences are explained by slipped pairing, nor are theyspontaneous hotspots. Because most of the 20 exceptionalmutants are inconsistent with slipped pairing models of mu-tagenesis, even rare mutations provide no evidence for 9-AAinduction of the slipped pairing mode of mutagenesis in T4.

Conclusions. These experiments implicate both the nucleaseand polymerase activities of T4 DNA polymerase in theprocessing of DNA nicks induced during 9-AA mutagenesis.This conclusion is based on our ability to correlate thespecificity of 9-AA-induced frameshifts (the deletion/duplication ratio) to the characteristics of the exonuclease andpolymnerase activities of the T4 DNA polymerase. The exper-iments do not rule out the occasional participation of addi-tional enzymes in nick processing. Clearly, multiple nucleasesmight make some contribution to deletion mutagenesis. In-deed, analysis of deletion/duplication specificity induced bytopo-active drugs in CHO cells suggests participation of othernucleases in nick processing in that system (26). In T4, it ispossible that E. coli DNA polymerase I might contribute toduplication mutations. The results presented here predict thatfurther studies using appropriate mutants would reveal thispossibility.The demonstration that the specificity of 9-AA-induced

frameshifts is altered in a manner consistent with predictions

based on the biochemical characteristics of T4 DNA poly-merase processing of topoisomerase nicks provides strongevidence that slipped pairing of DNA is not the molecularevent underlying frameshift mutagenesis induced by acridines.In addition, these studies provide a molecular perspective forunderstanding the diverse influences of mutant DNA poly-merases on mutational specificity. The L141 polymerase allelereduces certain classes of mutations, a characteristic routinelyattributed to improved proofreading by its highly active 3' -5' exonuclease (22, 27). However, other mutations, includingcertain frameshifts, are increased by this polymerase (refs. 9and 28; L.S.R., unpublished work). This study demonstratesone way that a highly active exonuclease might increasedeletion mutations.

We thank Dr. M. Masurekar for her advice and aid during the earlystages of this work. Zhi-Fang Chou performed preliminary 9-AAdose-response measurements. This work was supported by AmericanCancer Society Grant CN-50 to L.S.R.

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