MECHANISMS OF SPONTANEOUS AND INDUCED FRAMESHIFT MUTATION ... · MECHANISMS OF SPONTANEOUS AND INDUCED FRAMESHIFT MUTATION IN BACTERIOPHAGE T4 ... crease dramatically as a function

Copyright 0 1985 by the Genetics Society of Anierica

MECHANISMS O F SPONTANEOUS AND INDUCED FRAMESHIFT MUTATION IN BACTERIOPHAGE T4

GEORGE STREISINGER' AND JOYCE (EMRICH) OWEN'

Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Manuscript received October 22, 1984 Accepted November 17, 1984

ABSTRACT

Frequencies of spontaneous and proflavine-induced frameshift mutations increase dramatically as a function of the number of reiterated base pairs at each of two sites in the lysozyme gene of bacteriophage T4. At each site, proflavine induces addition mutations more frequently than deletion mutations. We con- firm that the steroidal diamine, irehdiamine A, induces frameshift addition mutations. At sites of reiterated bases, we propose that base pairing is misaligned adjacent to a gap. The misaligned configuration is stabilized by the stacking of mutagen molecules around the extrahelical base, forming a sandwich. Proflavine induces addition mutations efficiently at a site without any reiterated bases. Mutagenesis at such sites may be due to mutagen-induced stuttering of the replication complex.

UTATIONS in DNA that result in the addition or deletion of one or M more bases (BRENNER et al. 1961) are ubiquitous. In bacteriophage T4 they represent the most common spontaneous class, and they have been identified in bacteria (AMES and WHITFIELD 1966), yeast (STEWART and SHERMAN 1974) and humans (FITCH 1973). Because the addition or deletion of bases (except in multiples of three) results in a shift in the reading frame for the translation of the genetic message (CRICK et al. 196 l) , such mutations are often referred to as frameshifts.

Frameshift mutations occur with especially high frequencies in regions of repeated base sequences (OKADA et al. 1972; PRIBNOW et al. 1981; FARABAUCH et al. 1978). Previously (STREISINGER et al. 1966), we suggested a model that accounts for this observation; Figure 1 shows the steps we propose. Since frameshift mutations occur near ends of DNA molecules in T4 (DRAKE 1964; STRICINI 1965; LINDSTROM and DRAKE 1970), we indicate the creation of an end (a) and the subsequent digestion of one strand of the DNA molecule (c). If digestion stops adjacent to a stretch of reiterated bases, the melting of this stretch (d) could be followed by reannealing out of register, leaving one or more extrahelical bases bulged out of the duplex (e, f ) . When the undigested strand of DNA is used as template and the end of the complementary strand is used as primer, DNA synthesis restores a double-stranded molecule that is

' Deceased August 1 1 , 1984. Send reprint requests to: Institute of Molecular Biology, University of Oregon,

* Present addi-rss: 2830 Kinerald Sti-rec, Eugene, Oregon 97403. Eugene, Oregon 97403.

Genetics 109 633-659 April, 1985

634 G. STREISINGER AND J. (E.) OWEN

..... b. 1:

I I O I

. .... -- ..e.. -- g. - - h. _ _

I I I I I I I I I

I

FIGURE 1 .-lntermediates in the formation of frameshift mutations. a, Concatameric T 4 DNA molecule during intracellular growth, after endonucleolytic cleavage. 0 and vertical lines represent a sequence of reiterated base pairs. b, Formation of an end. c, Single-stranded exonuclease digestion. d , Melting of a short terminal stretch. e and f, Annealing in a misaligned position. g, Precursor to an addition mutation. h, Precursor to a deletion mutation.

identical with the original one except for the bulge defect. Note that, if the extrahelical base occurs in the primer strand (e), an addition will be generated, whereas a bulge defect in the template strand ( f ) results in a deletion. If no other events intervene, a subsequent round of replication generates one normal and one mutant molecule.

Bulge defects in which one or several bases extrude from an otherwise fully base-paired region are known to be sterically feasible (FRESCO and ALBERTS 1960). Extrahelical bases have been demonstrated directly in vitro by use of the appropriate model compounds (EVANS and MORGAN 1982). For single-base additions or deletions, a single extrahelical base is thermodynaniically more favorable (TINOCO et al. 1973) than are the extended unpaired regions we diagrammed earlier (STREISINGER et al. 1966). Here, we describe the frequencies of addition and deletion mutation at particular sites as a function of the

FRAMESHIFT MUTAGENESIS IN PHAGE T 4 635

length of the repeating stretch of bases. Our results complement studies of frequencies obtained at various sites in the rZZ gene (PRIBNOW et al. 1981). We compare our observations to expectations based on thermodynamic considerations.

I t has long been known that acridines increase the frequency of frameshift mutations (DEMARS 1953). Most models for acridine mutagenesis (LERMAN 1963; STREISINCER et al. 1966; LEE and TINOCO 1978) have invoked the intercalation of acridines between base pairs in the D N A , since intercalation is known to be a prominent mode of interaction. Such models are not entirely satisfactory since not all acridines that efficiently intercalate into DNA are strong mutagens (DRAKE 1970; ROTH 1974) and since mutagenesis occurs with higher-than-first-order kinetics (DRAKE 1970). Here, we describe further experiments that lead us to consider alternative models.

Some of the interpretations presented here were formulated some time ago (see DRAKE and BALTZ 1976). We have delayed presenting them in full partly for reasons described elsewhere (STREISINGER 1966) and partly because we hoped that a simple unified mechanism for frameshift mutagenesis would occur to us. Instead, it appears that several different mechanisms might operate, and a number of models remain in contention. As a result, parts of our discussion have evolved to resemble commentaries on a particularly ambiguous section of the Talmud.

MATERIALS AND METHODS

Bacteria, bacteriophage, media and mutant stock preparation: T h e bacterial strains Escherichia coli B (from S. E. LURIA), B phr- (from W. SAUERBIER) and KRA-8 (from N. ZINDER) and the bacteriophage strain T4B (from S. BENZER) were used. All bacteriophage stocks carry the mutations ac (SILVER 1967) and q (PIECHOWSKI and SUSMAN 1967) to facilitate growth in otherwise inhibitory concentrations of acridine.

Broth, buffered broth, bottom and top agar and citrate bottom agar were as described by OKADA et al. (1972). M-9 medium (ADAMS 1959) was supplemented with casamino acids (Difco) (5 mg/ml) and tryptophan (20 pglml). Citrate top agar contained 7 g/liter of agar, but otherwise was the same as citrate bottom agar.

Stocks of e mutant phage were prepared by plating 105-106 phage (obtained from a single 4- h r plaque) on B bacteria on citrate bottom agar (in glass Petri plates) with 2.5 ml of citrate top agar supplemented with 500 pg of egg-white lysozyme (Worthington). After 6 h r incubation at 37", plates with nearly confluent plaques were exposed to chloroform vapors and the top agar was scraped into 5 ml of broth, broken up with a 10-ml pipette and centrifuged at low speed to reniove agar and debris.

Irehdiamine A (IDA) mutagenesis and measurement of mutant frequencies: E. coli B bacteria were grown to a concentration of 1 X 1OS/ml in supplemented M-9, adjusted to 4 X 108/ml by centrifugation, aerated for 5 min at 37" and diluted 1:2 into phage suspensions to yield a multiplicity of infection of three to seven phage particles per cell. Five minutes after infection, cultures were diluted 1: 10 into flasks containing the appropriate IDA concentrations in supplemented M-9, shaken at 30" and lysed with chloroform and egg-white lysozyme at 60 min after infection. These experiments were performed in the dark. Stock solutions of IDA were prepared in 10% (v/v) aqueous ethanol and diluted to the appropriate concentration in M-9. Total phage were counted on citrate plates supplemented with egg-white lysozyme; e + phage were counted after plating with E. coli KRA-8 bacteria on citrate plates incubated at 43". T h e cultures treated with IDA had reduced burst sbes

636

Proflavine mutagenesis and measurement of mutant frequencies

Selective growth to remove preexisting wild-type or pseudowild-type phage: E. coli B were grown to a concentration of 1Os/ml in buffered broth, centrifuged in the cold, resuspended in buffered broth and adjusted to a concentration of 4 X 1OS/ml. The bacteria were then brought to 37", aerated and infected with a multiplicity of about five phage per bacterium. At 3 min after infection, antiphage serum was added to k = 10, and 3 min later the culture was diluted 1:4 in broth. At 22 min after infection, 10% (v/v) chloroform was added to lyse bacteria infected with wild-type or pseudowild-type phage, and the remaining (unlysed) bacteria (in the aqueous phase) were sedimented by centrifugation. The bacteria were washed twice with broth and were resuspended in broth. Chloroform was again added, followed by 10 &ml of egg-white lysozyme. The bacterial debris was sedimented by centrifugation, and the lysate was filtered through Celite (Johns-Manville and Company) to remove lysozyme and bacterial debris.

Growth for a single cycle in the presence or absence of mutagen: E. coli B were grown to a concentration of 3 X 108/ml in supplemented M-9 medium aerated at 37" and were infected with a multiplicity of three to seven phage particles per cell. Five minutes after infection, 8 rg/ml of proflavine (Nutritional Biochemical Corporation) were added if desired, the cultures were trans- ferred to 30" and the infected cells were superinfected 15 min after infection by the addition of three to seven additional phage particles per cell. Cultures not exposed to proflavine were lysed 45 min after infection with 10% (v/v) chloroform and 10 pg/ml of egg-white lysozyme, and bacterial debris was sedimented by centrifugation. At 60 min after infection, proflavine-treated cultures were sedimented and resuspended (in supplemented M-9 medium) to remove the proflavine. After a further 40-min incubation period at 30" they were lysed with chloroform and egg- white lysozyme and freed of debris by centrifugation. Treatment with proflavine and incubation of proflavine-treated phage were performed in the dark.

Measurement of the frequencies of wild-type or pseudowild-type phage present in the single-cycle lysate: When mutant lysates are plated directly under restrictive conditions, plaques may be formed not only by physiologically wild-type (or pseudowild-type) phage already present in the lysate, but also by new wild-type phage that arise during subsequent cycles of "leaky" growth of the parental phage on the Petri plate. Although these "plate revertants" vary in plaque size, in many strains they could not be distinguished unambiguously from preexisting revertants by inspection. The frequency of revertants was, therefore, measured by the single-burst technique (Ellis and Delbruck 1939). Revertant phage particles present in the lysates were identified as ones that gave rise to a full burst of progeny phage after a cycle of growth in bacteria distributed in large numbers of individual tubes. For these measurements, E. coli B were grown to a concentration of 108/ml in aerated buffered broth at 37" and then concentrated to 4 X 108/ml. After 5-min aeration at 37" they were infected with a low multiplicity of phage particles; 5 min after infection they were diluted ten-fold in broth at 34", and 0.05- to 0.25-ml amounts (depending on the experiment) were then distributed to single-burst tubes in a shaker at 34". A drop of chloroform was added to each tube 35 min after infection; after a few minutes for lysis, the contents of each tube were plated (including the chloroform) on citrate bottom and top agar and the plates were incubated at 43". The concentrations of infecting phage were adjusted so as to yield a mean number of fewer than 15 plate revertants per plate and to yield only a fraction of tubes with revertant bursts. Revertant bursts were defined as ones with more than 40 revertant (physiologically wild-type or pseudowild-type) phage. This cutoff was chosen to minimize the contributions of plate revertants and new revertants that arise early during the growth cycle of infected bacteria in single-burst tubes (LURIA 195 1) and to maximize the contributions of preexisting revertants. Reconstruction experiments suggested that only a minority of preexisting revertants would have been excluded, a fraction approximately equal to the fraction of new revertants that arise early during the infection cycle (LURIA 1951).

Direct platings were adequate for measuring induced mutation (reversion) in the eJ42 --f e+ and eJ42 + eJ42 eJ44 steps, because the frequencies of plaques formed by mutagenized phage were very high compared with those of untreated phage.

Identijication of mutants: Various genotypes that produced plaques under restrictive conditions were distinguished as follows. Plaques of e+ phage (in the eJ42 + e + step, for example) are surrounded by large halos after exposure to chloroform treatment and are readily distinguishable

G. STREISINGER AND J. (E.) OWEN

FRAMESHIFT MUTAGENESIS IN PHAGE T4 63 7

from plaques formed by pseudowild-type phage (for instance eJ42 eJ44 phage) (see figure 1 of STREISINGER et al. 1966). That these large-haloed plaques are indeed identical with e+ is suggested by the failure to find e (mutant-appearing) phage among progeny of crosses to the canonical e+ strain. To determine what fraction of the small-haloed phages among mutants were of the desired genotype (for instance, eJ44 eJ42 in measuring e+ + eJ42 frequencies), phage from independently arising plaques were tested genetically. T o extract single mutants, phage from stocks prepared from individual plaques were crossed to e+ phage, and the infected bacteria were irradiated with ultraviolet light (UV) in order to enhance recombination. The progenies of these crosses were subjected to selective growth to enrich for e phage and were plated under permissive conditions. T o determine the genotypes of the extracted e phage, they were grown into stocks and crossed to two strains, each carrying one of the mutations (for instance, eJ44 or eJ42) that are present in the pseudowild-type double-mutant strain. The presence of recombinants in one of the crosses (for instance, that to eJ44) and their absence in the other (for instance, to 4 4 2 ) define the presence of the mutant of interest in the particular strain. For all except the proflavine-induced e+ 4 eJ42 step, more than 80% of tested plaques proved to be of the indicated genotype, and corrections were not made for the small fraction of others. Only about 5 % of the proflavine-induced mutant bursts examined in the e+ --* eJ42 step proved to be of the desired genotype. The number of bursts listed for this step was calculated on the basis of 12 bursts identified as eJ42 eJ44, and the confidence limits are based on this number. The number of bursts indicated is larger than 12, because only one plaque was tested from each tube and a fraction of the tubes is calculated to contain several bursts.

The frequency of mutants: The mean number of bursts per tube was calculated from the fraction of tubes with no mutant bursts on the basis of the “0” term of the Poisson distribution (ADAMS 1959, pp. 485-488). As indicated earlier, mutant bursts are defined as ones with more than 40 plaques in order to distinguish preexisting mutants from ones arising during growth in the single- burst tubes.

RESULTS

The frequency of spontaneous mutation as a function of reiteration length: Ac- cording to the misaligned-pairing model, the frequency of mutation depends on the relative stability of the misaligned stretch of DNA, the likelihood of DNA synthesis using the misaligned primer and the probability of excision of the extrahelical base. To evaluate the influence of these factors on the frequency of frameshift mutation in bacteriophage T 4 , we measured the frequency of addition and deletion mutation at particular sites as a function of L, the maximum number of bases in the misaligned stretch. The stability of the misaligned stretch can be calculated on the basis of thermodynamic considerations: increasing L is expected to increase stability and, thus, to increase mutation frequency. Excision of the extrahelical base is expected to decrease the frequency of addition mutations and increase the frequency of deletions.

We have previously described a site in the wild-type T4 lysozyme gene with a sequence of five iterated base pairs (STREISINGER et al. 1966). Our attention was drawn to this site because it is the location of mutations with unusually high reversion frequencies (OKADA et al. 1972). Mutant strains with a base addition (eJDZI) (OKADA et al. 1972) or a base deletion (eJ42) (TERZAGHI et al. 1966) at this site have been isolated. The base sequences at the wild-type and mutant sites have determined (or predicted) and are described in Table 1.

According to the misaligned-pairing model, one possible intermediate in the

63% G. STREISINGER AND J. (E.) OWEN

TABLE 1

Base sequences at the eJ42-eJD11 and eK30-3J335 sites

Strain Base sequence

At eJ42-eJDZ Z eJ42 ' ,TTACAAAAGTCC..' e+ ..TTACAAAAAGTCC..b eJD I 1 ..TTACAAAAAAGTCC..C

At eK3U-eJ335 eK3U .,CAACAAAACGCT..' e + . . CAACAAAAACGCT . . b'r e1335 . .CAACAAAAAACGCT..d

Based on the comparison of amino acid sequences in e + and

Direct DNA sequence (OWEN et al. 1983). eJ42 eJ44 (TERZAGHI et al. 1966).

' Based on the comparison of amino acid sequences in e+ and eJDZU eJDZl (OKADA et al. 1972). The amino acid sequence . . . His-Leu-Leu-Gln. . . originally published for eJDlU eJDZZ (OKADA et al. 1972) must actually be . . . His-Leu-Leu-Leu-Gln. . . as determined by several independent sequenator analyses of related strains conducted for us by ROBERT BECKER at Oregon State University, Corvallis, Oregon. This alteration does not affect the eJDZZ sequence or any of the conclusions by OKADA et al.

Based on the comparison of amino acid sequences in e+ and eJ335 eJDlUl strains. The amino acid sequence at the relevant region in the e + strain is Gln-Lys-Arg-Trp-Asp-Glu, whereas in the eJ335 q1DIOI strain it is Gln-Lys-Thr-Leu-Asp-Glu, as established for us by K. WAUH and L. ERICCSON at the Department of Biochemistry, University of Washington, Seattle, using the method of HERMODSON et al. (1972).

( 1 972).

formation of the base addition mutation eJDl l from e+ has the following appearance:

A -- A-C A-A-A-A . . . . . .

---T-G-T-T-T-T-T-C-A---

I tL4 We vary L (the length of the paired but misaligned stretch of bases) by

examining different mutational steps at the eJ42-eJD11 site as follows:

4A (eJ42)

L = 3 JT L = 4

L = 4 JT L = 5

5A (e')

6A (eJDZ1)

The mutant frequencies observed for each of these steps are presented in Table 2. TWO key features are apparent. First, the frequency of spontaneous

FRAMESHIFT MUTAGENESIS IN PHAGE T4 639

TABLE 2

The frequency of spontaneous and induced frameshqt mutants

Frequency of niutants (X IO') after growth Factor o f increase

of mutant fre-

1. bulge defect Mutation mutation proflavine proflavine in proflavine Strand with Effect o f In absence of In presence of quency after growth

Primer Primer

Template Template

Primer Primer

Template Template

eJ42 + e+ e+ + eJDll e+ + eJ42 eJDl l + e+

eK30 + e+ e+ + eJ335 e+ --.* eK30 e1335 + e+

At eJ42-eJDI 1 site Addition 0.038" Addition 1.3 Deletion 4.6 Deletion 284

At eK30-eJ335 site Addition <0.023" Addition 3.5 Deletion 5.7 Deletion 105

92.4

21.7" 195

535

8.3

22.6 195

244

2400 150

4.7 1.9

360 56

4.0 2.3

See Table 3 for the specific steps examined and for the data. a These frequencies are based on small sample sizes; see Table 3 for confidence limits.

mutants increases as a function of L, for both additions and deletions. Second, for L = 4 (the only case for which a comparison is possible), the spontaneous addition and deletion mutation frequencies are similar, although deletions appear to be slightly more frequent than additions.

T o determine whether the mutation frequencies depend on L, rather than on features of the eJ42-eJD11 nucleotide neighbahood, idiosyncracies of the particular strains or the assay procedure, we examined a similar set of mutant frequencies at another site where a sequence of five A:T base pairs is present in the e+ strain (eK3O-eJ335, see Table 1). The observation that mutation frequencies are similar at both sites (Table 2) is compatible with the idea that the frequencies are mainly dictated by the sequence of repeating bases per se.

The following features of the experimental design (Tables 3-5) for measuring mutant frequency should be noted:

The reported mutant frequencies reflect mutations that occurred during one cycle of phage growth. Preexisting mutants were removed efficiently, and mutants arising during growth on the plate were distinguished unambiguously from ones arising during the one-step growth cycle by use of a modified single- burst method.

Some of the measured frequencies are for a change from a mutant to a wild-type sequence (e.g., eJ42 + e+), whereas others are for wild type to mutant. T o obviate the need for different modes of screening, all measurements were performed with populations that exhibit a mutant phenotype and with the mutation of interest resulting in phenotypically wild-type or pseudowild- type phage. These conditions, of course, already exist in the case of the step from a mutant (for instance, eJ42) to wild type ( e+) . For measuring the frequency of change of e+ to a particular mutant, we take advantage of the fact that particular double-mutant strains (carrying nearby addition and deletion mutations) produce nearly normal (pseudowild) lysozyme and produce plaques

640 G . STREISINGER AND J. (E.) OWEN

TABLE 3

Measurement of spontaneous mutant frequencies

No. of No. of phage Mutational Specific step tubes added No. of Frequency of

step nieasured examined (X lo-’) mutants niutants (X 10’)

eJ42 + e+ eJ42 + e+

e+ + eJD1 I eJD10 + eJDlU eJDl1

e+ + eJ42

eJDl l + e+ eJDll + e+

eJ44 + eJ42 eJ44

eK3U + e+ eK3U + e+

e+ + eJ335 eJDIUl + e5335 eJDlUl

At eJ42-eJD11 site 300 9.3 319 8.6 149 __ 3.0

20.9

270 3.8 3.5 7.3 - 220

936 11.5

180 0.026 199 0.047

0.073 -

At eK3U-eJ335 site 80 1.4

2.9 4.3 - 180

295 3.5 100 0.39 180 1.9

5.8 -

100 0.3 0.96 1.26 - 80

50 0.048 80 0.039

0.087

1 7 0 8

49.1 43.4 92.5

522.1

60.4 147.0 207.4

-

-

0 0 0

67.1 46.3 89.9

203.3

6.2 65.6 71.8

43.4 47.8 91.2

-

-

-

-

0.038 (0.016-0.08)

1.3

4.6

284

<0.023 (0-0.09)”

3.5

5.7

105 ~~ __ ~ ~ __

Mutant frequencies were measured in stocks from which preexisting wild-type or pseudowild phage were removed. Phage were grown in a single cycle, and the frequency of mutants was measured with the aid of a modified single-burst technique. The genotypes of the classes of interest were confirmed by genetic tests. More extended data are presented for one of the steps (e+ + eJ42) in Table 5.

a 95% confidence limits based on number of observed bursts.

and halos similar to those of wild-type phage. To determine the frequency of mutation from e+ to eJ42, for instance, we utilized eJ44 phage. The eJ44 strain carries a single-base addition 14 bases removed from the eJ42-eJD11 region, and the double-mutant strain eJ42 eJ44 is known to be pseudowild. Thus, mutation of the e+ sequence to the eJ42 sequence can be recognized easily if the phage already has the eJ44 mutation, since the resultant double mutant (eJ42 eJ44) appears pseudowild, whereas the parental eJ44 appears mutant. The identity of each mutant class of interest was established by genetic tests.

2 FRAMESHIFT MUTAGENESIS IN PHAGE T4

T A B L E 4

Mutant frequencies among phage grown in the presence of proflavine

No. of No. of single-burst No. of phage mutant

Mutational tubes plated or phage or Frequency of step examined added (X IO-’) bursts mutants (X 10’)

At eJ42-eJDI I site el42 + e+ 2.1 172

2.2 4.3 - 219

39 1 92

e+ -+ eJ42 229 200

eJDIl + e + 180

eK3U -+ e+

e+ -+ eJ335

0.074 25.0 0.1 1 10.8 0.18 35.8 195

-

0.55 9.5 0.42 0.97 - 11.4

20.9 21.7 (10.8-36.6)b -

0.31 165.5 0.1 1 63.5 0.43 229.0 535

At eK3O-eJ335 site 80 5.6 44.3

160 7.4 63.2 - 13.0 107.5 8.3

80 0.14 50.8 0.54 81.6 0.68 132.4 195 - 160 -

64 1

e+ + eK3U 150 2.85 85.0 3.44 57.1 6.29 142.1 22.6 - 160

eJ335 + e+ 100 0.18 49.5 28.5 - 0.14

0.32 78.0 244 - 80

Mutant frequencies were determined as described in the legend to Table 3, except that growth was in the presence of 8 pg/ml of proflavine.

Because the ratio of proflavine-induced to spontaneous mutations is high in this step, the induced mutants were measured by direct plating instead of by the single-burst method.

* 95% confidence limits based o n number of observed bursts.

Independent measurements of spontaneous mutant frequencies : A single-burst method was used to minimize the (often major) contribution of mutations that occur during the growth of phage after plating. For some steps the relative contribution of plate mutants was small when plaques were scored 6-7 hr after plating. To determine whether the single-burst method gave satisfactory estimates, we compared frequencies obtained by the two methods for two different steps. For e+ to eJDll (eJDl0 eJDII+ to eJDI0 e J D l l ) , the frequency observed


TABLE 5

Frequencies of spontaneous e+ + eJ42 mutants in independent stocks

Totdl no. of No. of infecting phage

single-burst No. of Mean no. Total no. added to single- tubes tubes with no of mutant of mutant burst tubes Frequency of

Stock examined niutant bursts bursts/tube bursts x 10-8 mutants x 10’

1 80 46 0.55 44.3 1 .0 4.4

2 60 26 0.84 50.2 0.78 6.4

3 77 48 0.47 36.4 1.2 3.0

4 80 48 0.51 40.9 1.4 2.9

5 99 50 0.68 67.6 0.62 10.9

6 80 73 0.09 7.3 0.57 1.3 80 35 0.83 66.1 1.2 5.5

7 100 85 0.16 16.3 0.68 2.4 100 45 0.80 79.7 1.6 5.0 100 55 0.60 59.8 1.6 3.7

6.7 - - - - 80 41 0.67 53.5 0.8

Total 936 522.1 11.45 Average 4.6

This table presents some of the data on which the calculation of mutation frequencies shown in Table 3 is based. The summarized data are shown in a single line in Table 3.

by direct plating was 2 X for the single-burst method, Tables 3 and 4); for e+ to eJ335 (eJD101 eJ335+ to eJDlOl eJ335) it was 5.5 X Tables 3 and 4). The single-burst method thus gives satisfactory estimates of mutant frequencies.

To measure mutation frequencies at the eJ42-eJD11 and eK30-eJ335 sites without depending on the use of double mutants, we isolated 102 independently arising spontaneous mutants and mapped them against a set of deletions by spot-test crosses. Mutations that occurred in the appropriate regions were crossed (with exposure to UV to enhance recombination) to eJ42 or eK3O: eight failed to recombine with eJ42 and another eight failed to recombine with eK30. None of these mutants exhibited a high reversion frequency; thus, we conclude that they are deletions of single bases and resemble eJ42 or eK30. The total spontaneous mutant frequency at the e gene is 3.7 X thus, the directly measured mutation frequencies from e+ to eJ42 or to eK30 are about 3 x a value that is within two-fold of the frequencies of 4.6 x and 5.7 X observed with our indirect method. The absence of addition mutations in this spontaneous set was surprising. As discussed later, we probably selected against them during the isolation procedure.

(compared to 1.3 X

(compared to 3.5 X


Of the remaining mutants in the set of 102, 27 mapped at or near two other sequences with five iterated base pairs (OWEN et al. 1983); thus, about 40% of the spontaneous mutations mapped at the four sites with the greatest reiteration length. The 20 base pairs at these sites occupy 4% of the entire DNA sequence of the lysozyme gene. Spontaneous mutation thus occurs with a relatively high frequency at sites of reiteration.

The frequency of projlavine-induced mutation as a function of reiteration length : The mutagenicity of acridines has been ascribed to their intercalating into, and stabilizing, double-helical stretches of DNA. If mutagenesis is due to intercalation within the misaligned stretch proposed in the bulge model, the frequency of induced addition and deletion mutation ought to be similar for any particular L, since the structure of the misaligned stretch is identical for both. T o test this prediction, we have measured the frequency of frameshift mutation after phage growth in the presence of proflavine (Table 2). A striking result is that the frequencies of addition mutation are enhanced much more than the frequencies of deletion mutation. For example, the frequency of proflavine-induced addition mutation with L = 4 is almost ten-fold higher than the frequency of deletion mutation with the same L. This result does not lend support to the notion that proflavine mutagenesis is due to intercalation within the misaligned stretch of DNA. Inequalities between the frequency of addition and deletion mutation have been described also at the l a d gene in E. coli (CALOS and MILLER 1981) and will be discussed later.

The mutagenicity of a steroidal diamine: It had been reported earlier that the steroidal diamine pregn-5-ene3/3,20a diamine (IDA) increases the frequency of revertants of rZZ frameshift, but not transition, mutants (MAHLER and BAYLOR 1967). IDA intercalates only into kinked regions of DNA (SOBELL et al. 1977) and, at higher concentrations, is known to destabilize double-stranded DNA (MAHLER et al. 1966). As is shown in Figure 2, we have confirmed the mutagenicity of IDA by finding a dose-dependent increase in the frequency of eJ42 + e+ addition mutations. As is the case for acridines, mutation is induced with higher-than-first-order kinetics: the slopes of the curves that relate log (frequency of mutants) to log (dose) range from 3.7 to 5.7 in the three different experiments summarized in Figure 2. As with acridines (DRAKE 1970), the frequency of mutants levels off or decreases at supraoptimal concentrations of mutagen. However, the mutagenic potency of IDA for the eJ42 + e+ step is considerably lower than that of compounds such as proflavine.

We found no appreciable increase in the frequency of deletion mutation in the presence of IDA and no increase in the frequency of transition mutations (data not shown). The eJ44 site will be described.

The frequency of induced mutation at a nonreiterated site: Some proflavine- induced mutations occur at sites without a reiterated sequence of bases (STREIS- INGER et al. 1966). T o determine whether such mutations occur often, we measured the frequency of proflavine-induced mutation at the eJ44 site (Table 6), where a single G:C base pair is added adjacent to a single resident G:C base pair:

644

>I C aJ 0

CL e

h 0 7

L aJ

ln C 0

3

a

44

&

2


10.0

5.0

2.0

1 .o

0.5

I

I I I I I I I I

50 100 200

I D A (pM)

FIGURE 2.--IDA mutagenesis. The results of three different experiments are plotted. The frequency of spontaneous mutants has been subtracted from the points shown. Slopes were calculated from the points connected by lines. The variability with eJ42 is probably due to concentration differences that arose unwittingly during the preparation of stock solutions from very small amounts of mutagen. Plating conditions are described in Table 6. A and W, The frequency of eJ42 + e+ addition mutants. 0, The frequency of eJ44 + e+ mutants.

--- A - T a - T - - - e'

4 --- A-T-G-G-C-T--- eJ44

These mutations were measured at the same time as were the frequencies of mutation from eJ42 to e+ (Table 4). In addition to plaques with large halos (formed by e+ phage), plaques with smaller halos were observed that were indistinguishable from those formed by eJ42 eJ44 phage; the genotypes of phage from .six independently arising small-haloed (pseudowild) plaques were established as eJ42 eJ44 by the appropriate crosses. It is striking that the frequency of proflavine-induced mutation at the eJ44 site, 5.7 X (Table 6), is higher than that for any induced mutation at the stretches of reiterated A T base pairs we have examined.

T o determine whether the high mutability of the eJ44 site was due to the immediately adjacent base sequence, we measured induced mutation frequen-


TABLE 6

Comparison of mutant frequencies at the eJ42 and eJ44 sites

eJ42 -+ e' e+ -+ eJ44

Conditions

Number of phage No. of Frequency plated or added niutant phage of mutants

(X 10-7) or bursts (X 107)

No. of mutant phage Frequency of

or bursts mutants (X IO')

Direct plating Spontaneous" Proflavine' IDA" (120 pM)

Single burst Spontaneous' IDA (75 pM)

240 65 0.32 4.2 39 1 92.4 7.5 35 4 .3

210 8 0.04 13 13 1 .0

15 0.07

6 0.8 2,386 568

7 0.03 3 0.23

a Plating was on KRA-8 bacteria on citrate plates incubated at 43". These conditions minimize

' Sorne of these results were also shown in Table 3. revertants that arise on plates.

cies at a site with the same base neighborhood (e+ to eJD22 at base pair number 360, OWEN et al. 1983):

--_ A-T4-C-T--- e+

.1 __- A-T-C-G-C-T--- eJD22

The frequency of induced mutants at this site was no greater than 4 X lo-' as judged by the frequency of pseudowild (possibly eK30 eJD22) plaques in a mutagenized eK30 population. The spontaneous mutant frequency was (probably much) less than 1 X lo-'. (The genotypes of the pseudowild plaques were not investigated.) Thus, the frequency of proflavine-induced mutations at this second nonreiterated site was more than 100-fold lower than at the eJ44 site. We conclude that the high frequency of induced mutation at the eJ44 site is not due exclusively to the immediately adjacent base sequence.

To further compare mutagenesis at the eJ44 and eJ42 sites, we measured the frequency of IDA-induced eJ44 mutants (Table 5 ) . As with proflavine, e+ + eJ44 addition mutation was detected in the same strain (eJ42) as was eJ42 + e+ addition, the two being distinguished by halo size. In contrast to proflavine, which was five times more potent in inducing e+ - eJ44 as eJ42 + e+, IDA was more than five-fold less efficient for e+ + eJ44 than for eJ42 --j e+. Thus, the relative potencies of the two mutagens are very different at the reiterated and nonreiterated sites.

DISCUSSION

Spontaneous frameshijt mutation Conditions of measurement and the comparison of frequencies at the e and rII

genes: To achieve physiologically similar conditions for the mesurement of all mutation frequencies, we measured the frequency of forward mutations in


strains that already carried mutations. For example, the e+ + eJDl1 frequency was measured by examining the step eJDl0 e J D l l + --* eJDl0 eJDl l and the e + ---* eJ42 frequency was measured by examining the step eJ44 eJ42+ + eJ44 eJ42. To determine whether the frequencies we measured were perturbed by the indirect methodology, we also measured mutation frequencies directly in a set of spontaneous mutants. The two approaches yielded frequencies that differed by less than a factor of 2 for the e + --* eJ42 and e+ + eJ335 steps, a remarkable agreement considering the different methods used.

Among the 16 mutants isolated at these two sites by the direct method none originated by base pair addition; on the basis of the frequencies observed with the indirect method, about eight addition mutations would have been expected. It is probable that eJDll-l ike and eJ335-like mutants were not re- covered because their plaques are less distinctly mutant in appearance than plaques of eJ42 or eK30. The reason for indistinctly mutant plaques is most likely to be the high revision frequency that results in the formation of e + revertants that are strongly selected even under the “permissive” plating conditions used for mutants.

Although mutability at iterated-A sites has been studied at the rZZ as well as the e locus, comparisons are sometimes uncertain because of both methodolog- ical and intrinsic differences. In the e system, we first sharply reduce the spontaneous mutant (or revertant) background of a stock by a round of selective growth and then measure new mutations arising in a subsequent round of nonselective growth; under these conditions, mutation frequencies should be similar to mutation rates (LURIA 1951; DRAKE 1970). In the rZZ system, mutation frequencies are usually determined using high-titer stocks grown under selectively neutral conditions (J. W. DRAKE, personal communication). The two wild-type sites studied in the e system each contain five As, and mutation rates for similar changes in L are similar at the two sites. The three wild-type sites studied in the rZZ system each contain six As, but mutation rates for similar change in L exhibit an approximately ten-fold range among the three sites (PRIBNOW et al. 1981).

Despite these differences, mutation frequencies exhibit large increases in both systems with increasing values of L.

In the e system, deletions and additions arise at similar frequencies from a site of L = 4 (with a 2.1-fold bias in favor of deletions). In the rZZ system, when L = 5, deletions and additions arise at similar frequencies at the r l 3 l site (with a 1.4-fold bias in favor of deletions) (BENZER 1957) but are reported by PRIBNOW et al. to arise at the r117 site with a 100-fold bias in favor of deletions. However, this latter bias may have been created by the misclassifi- cation of r117 addition mutants as rZZZ mutants because of their extremely high (ca. 0.1%) frequencies of revertants; indeed, in a deliberate search for rapidly reverting rll mutants, more were found at r117 than at r131 (J. W. DRAKE, personal communication).

Mutation frequencies from five to six As are 1.3 and 3.5 X lop7 in e and are 0.3, 1.9 and 6.1 X in rZZ. Equation 5-10 from Drake (1970) may be used to calculate the corresponding mutation rates: m = 0.4343 (f - fo)/log(N/No), where m is the mutation rate, f and fo are the final and initial mutation fre-


quencies, respectively, and N and No are the final and initial total population sizes, respectively. For the e system, it is appropriate to set fo = 0 and to take the burst size, approximately 200, as the value for N/No; the corresponding mutation rates are 2.5 and 6.6 X For the rZZ system, fo = 0, N is typically about 10" and it is appropriate to take No = m-' since mutants do not begin to appear in a population until it reaches a size that is the reciprocal of the mutation rate; the corresponding mutation rates are 0.4, 1.9 and 5.6 X Thus, the higher two rlZ rates are indistinquishable from the two e rates. If we assume that deletions at the e sites occur 2.1-fold more frequently than additions for L = 5 (as actually observed for L = 4), then averaged total mutation frequencies at six-A e sites would be about 3 X Observed mutation frequencies from the three six-A rZZ sites (to both five and seven As) are about 1.6, 8.7 and 16 X (PRIBNOW et al. 1981), values approximately as expected if the mean estimated e value were taken as a standard and corrections were made for different methodologies. Further comparisons of our e values and the rZZ values tabulated by PRIBNOW et al. yield both concordant and discordant results.

Taken as a whole, the e and rZZ results are very similar and, when different, may signal effects of nearby base sequences such as those seen with eJ44 vs. eJD22.

Mutation at regions of reiterated bases: Spontaneous frameshift mutations that occur with a high frequency are often observed in reiterated sequences in the l a d gene of E. coli (FARABAUGH et al. 1978), the rZZ gene of phage T 4 (PRIB- NOW et al. 1981) and the lysozyme gene as described here. In each case, the added sequences reiterate adjacent ones already present. These observations are compatible with the misaligned-pairing model.

An interesting alternative model for spontaneous frameshift mutation invokes events occurring at quasipalindromic sequences (RIPLEY 1982). This model predicts neither the reiteration of adjacent sequences nor the frequency dependence on L and is thus excluded for the sites described here, although it does account for some otherwise inexplicable frameshift mutations in yeast and in the rZZ gene of phage T4. RIPLEY, GLICKMAN and SHOEMAKER (1983) found that mutant DNA polymerases have different effects on frameshift mutation rates at reiterated and at quasipalindromic sites, enhancing the latter but reducing the former and thus further indicating the operation of multiple mechanisms of frameshift mutagenesis.

Frequency of mutation as a function of L: T o determine whether the large increases in mutation frequency observed with increasing values of L can be explained by thermodynamic considerations alone, we calculated the effect of varying L on the thermodynamic equilibria between misaligned and normally paired stretches. (Remember that L is one less than, not equal to, the number of base pairs in an iterated run.) In the misaligned-pairing model, for instance, the intermediate in deletion mutation differs from completely base-paired DNA by an extrahelical base which contributes a certain free energy loss (AG) for any one particular base that is bulged; the equilibrium frequency for any such mutational intermediate is exp(-AG/RT) as long as the misaligned intermediate is rare. For the total equilibrium frequency, this value must be multiplied


by the number of positions at which bases could be bulged out to yield a deletion mutation intermediate. For L = 5, a bulge defect could be formed at any of five positions (each still leaving the terminal base paired), whereas for L = 4 any of four bases could be bulged. The expected increase in the frequency of frameshift mutation as L increases from 4 to 5 is thus a factor of 5/4, or 1.25. The observed factors of increase are about 20 and 60 at the two sites examined (e+ + eJ42 compared to eJDl1 + e’ and e+ + eK3O compared to eJ335 + e + , Table 2). For addition mutation, as L increases from 3 to 4, the factor of increase expected is 413, or about 1.3, whereas factors of about 20 and more than 150 were observed at the two sites described in Table 2. In the rZZ region, large differences in mutation frequency were also observed at sites that differed in the number of reiterated bases (PRIBNOW et al. 1981). Thus, simple equilibrium considerations alone cannot explain the differences in mutation frequency.

At least two other factors could contribute to the large increase in mutation frequency as L increases. With longer L there would be an increase in the stability of the misaligned stretch and, thus, a greater opportunity for replication while in the misaligned state. Another possibility is the occurrence of steric interactions between the extrahelical base and the DNA replication complex. The DNA polymerase (gene 43 protein), for instance, has been proposed to make contact with four bases at the 3’OH end of the primer strand (NEW- PORT et al. 1981). As L increases, the bulge defect would be located at a greater distance from this end and would offer less steric hindrance to replication initiated on the misaligned primer stretch.

Addition compared to deletion : Before discussing further the relative frequencies of additions and deletions, it is profitable to consider the set of structures that we propose as intermediates in the formation of mutants:

A .1 (1) 5’---A-C A-A-A-A . . . . . .

3’---T-G-T-T-T-T-T-C-A---

(2) 5’---A-C-A-A-A-A-A-G-T--- . . . . . . T-T-T-T C-A---

T \/ t

.1 (3) 5‘---A-C-A-A-A-A . . . . . .

3’---T-C, T-T-T-T-C-A---

\ / T

A (4) 5’---A-C-A-A-A-A G-T--- . . . . . .

T-T-T-T-C-A---

t


Structures 1 and 2 are putative intermediates for the addition of an A:T base pair to a stretch of five A:T pairs (as in the e+ lysozyme gene at the eJDl1-eJ42 site), whereas structures 3 and 4 are intermediates in the deletion of a single base pair.The next step in the process of mutation is polymerization, by the addition of bases to the 3’ ends of the primer strand, at the positions shown by arrows (see also the step illustrated in Figure 1, e to g or f to h).

The following is an alternate set of possible intermediates:

/”\ (8) 5‘---A-C A-A-A-A-G-T--- . . . . . .

3’---T-G-T-T-T-T

In bacteriophage T 4 we consider structures 5-8 to be less likely candidates for mutational intermediates than 1-4. This is because, in T4, frameshift mutations arise near ends of DNA molecules, and the single-stranded regions in the mutational intermediates are likely to extend from the site of the mutation to the end of the molecule (see Figure IC). Under these conditions, DNA synthesis in structures 1-4 could proceed directly from the 3’ end of the primer strand, whereas for structures 5-8, independent priming and subsequent ligation would be required.

The relative frequencies of spontaneous addition and deletion mutation are of special interest since, as described in the next section, acridines are more potent inducers of additions than of deletions. The structures shown above suggest several sources of asymmetry between the frequencies of addition and deletion mutation. Before considering these, we calculate the relative frequencies of additions and deletions expected from equilibrium considerations. For deletions, the misaligned intermediate differs from the normally base-paired form merely in having a bulge defect, as we have already described. In contrast, for additions, a bulge defect is present and, in addition, a base pair is lost:


bulge base pair I lost

4 /”\ __- A-C A-A-A-A

J. addition __- A-C-A-A-A-A . . . . . .

--- T-G T-T-T-T-C-A--- \ / T

I deletion

The free energy difference (AG) due to the lbss of a single-base pair (in additions as compared to deletions) is about 1.3 kcal/m (TINOCO et al., 1973), and the difference between the two equilibria, exp(-AG/RT), is a factor of about 5. As shown in Table 2, with L = 4, deletion mutation frequencies were two to four times greater than addition mutation frequencies, not very different from the factor of 5 expected from thermodynamic equilibrium considerations. The small departure of experimental observations from thermodynamic expectations could be due to the variability inherent in our experiments. An- other possible explanation is that a bulge defect on the template strand (as it occurs in a deletion mutation) offers less steric hindrance to the polymerase than a bulge defect on the primer strand.

The consequences of repair: We assume that any repair systems that may operate will be specific and first consider ones that act by removing an extrahelical base. In addition mutation heterozygous intermediates (as diagrammed above), the excision of the extrahelical base (followed by ligation) would result in normal, unmutated duplexes; thus, the frequency of spontaneous addition mutants would be decreased. In deletion mutation heterozygous intermediates, in contrast, excision of the extrahelical base would result in a homozygous mutant duplex; thus, the frequency of spontaneous deletion mutants would be increased up to two-fold.

In bacteriophage T4, frameshift mutation recombinational heterozygotes (created in mixed infections of normal and mutant phage) do not seem to be repaired with particularly high efficiencies (LINDSTROM and DRAKE 1970), and repair within the helix (subsequent to the formation of the mutant stretch) may not perturb frameshift mutation frequencies to an appreciable extent. In organisms with efficient repair, we predict that the frequency of spontaneous addition mutations relative to deletions will be unequal: preferential removal of the extrahelical base will render deletions more frequent than additions. However, repair systems in some organisms may act by breaking the phospho- diester bond opposite the bulge. In such organisms, additions would be more frequent than deletions.

Repair could also occur at the site of DNA synthesis, due to the 3’-exonuclease editing function of the polymerase which results in turnover of the

FRAMESHIFT MUTAGENESIS IN PHAGE T4 65 1

terminal base on the primer strand. This repair would occur particularly efficiently where the bulge migrates to the end of the primer strand; its frequency would thus be a function of L. Since turnover occurs only on the primer strand, this mechanism may act preferentially to remove potential addition mutations.

Unequal crossing over: It has been suggested that addition and deletion mutation is the result of unequal crossing over between normal parental DNA molecules. To account for the single-base addition and deletion mutation discussed here, intermediate structures would appear as follows: --- W --X -A-A-A-A-A Y -Z --- --- W’-X’ T-T-T-T-T-y’-Z’---

--- W-X-A-A-A-A z --- --- W‘ T-T-T-T-y’-Z’---

where roman and italic letters represent contributions from two different parental DNA molecules. Base addition and ligation would complete the mutation.

Unequal crossing over is an unlikely source of frameshift mutation in T 4 for reasons summarized by LINDSTROM and DRAKE (1 970): newly arisen frameshift mutants are not recombinant for flanking markers, and the frequency of mutation is not depressed under conditions that reduce recombination, or stimulated by conditions that increase recombination. The unequal crossing over model also fails to account for features of induced mutation which will be discussed. Finally, recombination frequencies observed with short regions ( 1 5-30 base pairs) of homology (BAUTZ and BAUTZ 1976; SINGER et al. 1982) are much lower than we describe here for frameshift mutation with L = 4-5. Induced frameshift mutation

Possible sites of interaction of mutagen and models of frameshift mutagenesis: Mutagenic interactions could fall into two categories. In one, the mutagen is assumed to stabilize a previously misaligned configuration of DNA; in the other the mutagen causes the misalignment. The two types of interaction are diagrammed in Figure 3.

Stabilization of a misaligned region has been suggested to occur by intercalation within the misalignment (LERMAN 1963; STREISINGER et al. 1966) (site A, Figure 3) or opposite the extrahelical base (LEE and TINOCO 1978) (site B, Figure 3). We now propose that stabilization could occur by the stacking of mutagens on one or both sides of the extrahelical base, in the latter case forming a sandwich (site C, Figure 3).

Alternatively, interaction of mutagen with the primer strand ahead of the point of DNA synthesis (site D, Figure 3) has been suggested to cause stuttering of the replication complex, with resultant recopying of an already copied base (SAKORE, REDDY and SOBELL 1979). Another possibility that has not been considered sufficiently is that mutagen may become associated with protein components of the replication complex, causing it to lose precision -(ROTH 1974).


0 A A

c c I I --*, - - , I ; , ; , - -

FIGURE S.-Possible positions of association between mutagen and DNA. A, Intercalated within the duplex. B, Intercalated opposite an extrahelical base. C, Stacked on an extrahelical base. D, Associated with a single-stranded region.

We have been unable to account for all features of mutagen-induced frameshift mutation by invoking a single category of interactions. Models that invoke the stabilization of a misaligned stretch account for mutagenesis observed at reiterated stretches of base pairs but do not explain the addition of single base pairs at noniterated sequences. Models that account for this latter class of mutations do not explain the observed length dependence of mutation frequencies at reiterated bases.

Mutagenesis at reiterated sites: Models of induced frameshift mutagenesis at reiterated bases in bacteriophage T 4 are challenged by the following observations:

The frequency of mutation is increased in the presence of mutagen. The added base or bases reiterate adjacent sequences. The frequency of mutation is a function of L, the number of bases in a

misaligned stretch. Increasing L by a single base results in up to a 15-fold increase in mutation frequency.

Induced additions are more frequent than induced deletions for a given value of L.

A bulky molecule such as IDA can be a frameshift mutagen. Some efficient intercalators (such as 1 0-methylacridinium) are poor muta-

Mutagenesis follows higher-than-first-order kinetics. The stabilization models offered heretofore do not meet these challenges to

our satisfaction. Intercalation : That acridines stabilize DNA helices by intercalation is well

known. LERMAN (1963) first suggested that intercalation leads to mutagenesis, and STREISINGER et al. (1 966) suggested that intercalation stabilizes misaligned stretches. This model is unsatisfactory. It does not explain the lack of mutagenicity of some efficient intercalators, the mutagenicity of IDA [which is known to destabilize rather than stabilize DNA helices (MAHLER et al. 1966)], the higher frequency of induced additions relative to deletions or the selective stabilization of misaligned stretches.

gens.


A modified version of the intercalation model invokes the formation of charge transfer complexes (SCHREIBER and DAUNE 1974). The model is incom- patible with the high frequency of mutation observed at stretches of A:T base pairs, which cannot form charge transfer complexes, and with the mutagenicity of acridine orange (ORGEL and BRENNER 1961) and IDA, neither of which can form charge transfer complexes. G. STREISINGER and P. MCCABE (unpub- lished results) have confirmed the mutagenicity of acridine orange under conditions that exclude photodynamic activation.

Intercalation opposite an extrahelical base: Ethidium intercalates opposite an extrahelical base in an interaction that has been suggested to give rise to mutation (LEE and TINOCO 1978). The higher frequency of addition than deletion can be explained by this model only if it is assumed that selectivity exists with respect to the extrahelical base (see discussion that follows). The higher-than-first-order dependence of mutation on mutagen concentration could be explained only by a secondary mechanism such as the induction of enzymes needed for mutagenesis. Although these difficulties may not be fatal for this intercalation model, they sharply diminish its attractiveness.

The sandwich model: The stacking of mutagen around or next to the extrahelical base, forming a sandwich (either regular or open-faced) with the base as the filling, avoids some of the difficulties of the previous models. Mutagen- esis is due to the stabilization of the extrahelical base by stacking interactions with mutagens. The bulk of IDA does not interfere with stacking. The higher- than-first-order dependence of mutagen concentration is due to the participation of several mutagen molecules in the sandwich, including noninteger av- erages. The higher frequency of additions compared to deletions can be explained in the following three different ways.

Acridines stacked along the template strand may offer steric hindrance to the polymerase: We described earlier the observation that the ratio of spontaneous addition mutations to deletions is somewhat higher than would be expected on thermodynamic grounds. A possible explanation is that an extrahelical base on the template strand offers more steric hindrance to the polymerase than does an extrahelical base on the primer strand. An inspection of models makes it obvious that the bulges in the two positions occupy very different positions relative to the end of the primer strand. The observed greater frequency of induced addition mutation could be a reflection of such a difference in steric hindrance: for deletions, the stabilizing effect of acridines on the extrahelical base on the template strand could be partially negated by the increase in steric hindrance due to the added bulk of the stacked acridine. If we assume that the steric hindrance due to the added acridines is less for an extrahelical base on the primer strand, the frequency of addition mutations will be relatively greater than that of deletions, as is observed.

Acridines protect addition mutations against repair: Acridines stacked around an extrahelical base would be expected not only to stabilize it but perhaps also to protect it against repair. Repair systems that remove the extrahelical base would decrease the frequency of additions but increase the frequency of deletions; protecting against repair would thus result in an increase of additions

654 G . STREISINGER AND J. (E.) OWEN

relative to deletions. As pointed out earlier, the similarity of the frequency of spontaneous addition and deletion mutation in the e and rII genes argues against such a repair system in T4. Repair would help explain the T 4 results only if two gratuitous assumptions are made: the frequency of formation of spontaneous addition mutation intermediates would have to be much higher than that of deletion mutation intermediates, and an efficient repair system would have to be assumed to remove the bulk of spontaneously formed extrahelical bases, resulting in fortuitously similar spontaneous deletion and addition frequencies. There is little support for these assumptions: it is difficult to imagine why addition mutation intermediates should be formed much more frequently than deletion ones, and the repair of frameshift mutation heterozygotes seems to be no more efficient than the repair of transition mutation heterozygotes (LINDSTROM and DRAKE 1970).

Ends of DNA molecules in bacteriophage T4 may not be distributed randomly: We imagine that the first step in the formation of bacteriophage T 4 frameshift mutations is the generation of an end. We are driven to this assumption because of the observations that newly arisen frameshift mutations are found in regions of terminal redundancy in mature phage particles (DRAKE 1964; STRI- GINI 1965; LINDSTROM and DRAKE 1970). Cuts that generate ends of T 4 DNA molecules may not occur randomly. T o appreciate the consequences of preferential cutting sites, consider the mutagenic intermediates 1 through 4 diagrammed in the section on spontaneous mutation. If cuts occur more frequently to the right than to the left of this particular sequence, then structures 1 and 3 would be more frequent than 2 and 4. It is known that some acridines stack more efficiently around purines than around pyrimidines (SAKORE et al. 1977), and, thus, structure 1 (addition) would be stabilized more efficiently than structure 3 (deletion), giving rise to more induced additions than deletions. Similar explanations could be applied to other organisms if nicks (that generate gaps) are not randomly distributed. The application of this model to frameshift mutagenesis at the l a d gene of E. coli will be discussed.

Further tests would be needed to determine whether the sandwich model explains the observed hierarchy of mutagenic potency of various acridines.

Induced frameshiji mutation at the lac1 gene of E. coli: ICR-191-induced mutations in the l a d gene of E. coli are preponderantly additions or deletions of a single G:C base pair at sequences of three (and in one case four) reiterated G:C base pairs (CALOS and MILLER 1981). The frequent induction of mutations at sites of reiterated bases and the dependence of mutation frequency on L are similar at the l a d gene of E. coli and the e gene of T4.

In the lacl gene, addition mutations at some sites were found to be more frequent than deletions; at other sites the reverse was true. According to CALOS and MILLER (1981), “the preference for +1 frameshifts or -1 frameshifts is site specific, so that at some sites +1 frameshifts predominate by a 10: 1 ratio, whereas at other sites -1 frameshifts are favored by an approximately 2:l ratio.”

We believe that ratios of additions to deletions can be interpreted to obey simple region-specific rules that follow from one of the models we have dis-

+-

60-

50-

40-

30-

20-

10-

0-

10-

FRAMESHIFT MUTAGENESIS IN PHAGE T4

a

I I I a

I a

0 0

I ! I I I I

0 0 0

a

20

655

a 300 100 200

cussed. In any given region within the lacl gene, additions will be more frequent than deletions if the stretch of reiterated G bases is on one particular strand. Conversely, within the same region, deletions will be more frequent than additions if the stretch of G bases is on the opposite strand. A reversal of these rules defines a new region. This is illustrated in Figure 4 (adapted from figure 8 of CALOS and MILLER 1981), where we symbolize the positions of reiterated G bases by circles: a circle above any bar indicates that the stretch of G bases is on the top strand of the DNA (as drawn by FARABAUGH 1978); a circle below the bar indicates that it is on the bottom strand. Filled circles indicate regions in which a stretch of G bases on the top strand favors additions and a stretch of G bases on the bottom strand favors deletions; empty circles indicate regions where the opposite is true. These rules define one region with eight sites, one with four, one with three and two each with one site. Run analysis (SOKAL and ROHLF 1969) suggests that the probability is less than 0.05 that this grouping of sites is random.

An explanation presented before for the higher frequency of additions compared to deletions in the e gene can be modified to account for the observations at the l ad gene. We assume that ICR-191 interacts more readily with one of the two bases, G or C, and that gaps, which are precursors to mutation, are not distributed randomly. If the interaction is preferentially with G, for example, then structures a and c illustrated below would lead to mutation more


frequently than structures b and d. a b

. . . . __--- c-c-c __-_ -____ G-G-G ___-

C G-G _____ d c-c _____ . . . .

___ C,-G --__ --- c-c---- \ I

C \ I G

Thus, a gap in the top strand, near a reiterated sequence of G bases in the same strand, would lead to an addition (a), whereas the same gap near a stretch of C bases would lead to a deletion (c). A gap in the bottom strand (not shown) would have the opposite consequences.

We suggest that each of the regions identified in the lacZ gene is distinguished by the preferential formation of a gap in a particular one of the two strands of DNA, perhaps originating through a nick that is cut preferentially at a particular sequence. The nick would then be extended by nucleolytic digestion. In any one region, all sites could mutate next to gaps that originate from the same nick.

The abrupt transitions observed at the border regions are not predicted by our suggestion and pose a difficulty. A simple, although gratuitous, explanation could be a barrier to nucleolytic digestion (due perhaps to secondary structure) that defines region boundaries. Our explanation for the ratios of addition to deletion could be tested by substituting a sequence of reiterated Gs for a sequence of Cs at a particular mutable site. Such substitutions might be possible at mutable site 5 of the ZacZ gene (CALOS and MILLER 1981) with minimal perturbations of amino acid sequence (FARABAUGH 1978).

Mutagenesis at nonreiterated sites: Misaligned pairing requires reiterated sequences. Induced mutations at sites such as eJ44 cannot depend on the stabilization of misaligned sequences because, prior to mutation, no reiterated sequences exist. SAKORE, REDDY and SOBELL (1979) suggest that acridines may remain transiently stacked among bases in recently created single-stranded regions, and that mutations are caused by acridine-induced slippage of the primer-associated replication complex that results in looped-out stretches. The specificity of forming mutations at some, but not other, sequences that resemble those at eJ44 remains to be explained, perhaps by neighboring sequences that favor intercalation.

The differential response to the mutagens proflavine and IDA at a reiterated and nonreiterated site is compatible with the Sakore, Reddy and Sobell model: IDA stacking around an extrahelical base could occur more readily than IDA intercalation.

This mechanism does not explain the observed dependence of induced mutation frequency on L at sites of repeated bases. For these sites, the sandwich model or its analogues must still be invoked.


Thanks are due to YVONNE MAYNARD for preparation of lysozyme for sequence analysis, to SUE GRACE for help with single-burst experiments, to the now unknown person who supplied G.S. with the sample of IDA, to KEN WALSH, LOWELL ERICSON and BOB BECKER for sequenator analysis of mutant lysozymes and to FRANK STAHL and JAN DRAKE for extensive help in the preparation of this manuscript. Most of all, J.O. thanks GEORGE STREISINGER, a truly great scientist, whose imagination and insight in theory and experiment were equaled only by his procrastination in writing papers. This work was supported by United States Public Health Service grant GM 19795 to G.S.

LITERATURE CITED

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AMES, B. N. and H. J. WHITFIELD, JR., 1966

BAUTZ, F. A. and E. K. F. BAUTZ, 1967

BENZER, S., 1957

BRENNER, S., L. BARNETT, F. H. C. CRICK and A. ORGEL, 1961

CALOS, M. P. and J. H. MILLER, 1981

CRICK, F. H. C., L. BARNETT, S. BRENNER and R. J. WATTS-TOBIN, 1961

DEMARS, R. I., 1953

DRAKE, J. W., 1964

DRAKE, J. W., 1970

DRAKE, J. W. and R. H. BALTZ, 1976 The biochemistry of mutagenesis. Annu. Rev. Biochem. 45: 11-37.

ELLIS, E. L. and M. DELBRUCK, 1939 The growth of bacteriophage. J. Gen. Physiol. 22: 365-

EVANS, D. H. and A. R. MORGAN, 1982 Extrahelical bases in duplex DNA. J. Mol. Biol. 160:

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MECHANISMS OF SPONTANEOUS AND INDUCED FRAMESHIFT MUTATION ... · MECHANISMS OF SPONTANEOUS AND INDUCED FRAMESHIFT MUTATION IN BACTERIOPHAGE T4 ... crease dramatically as a function