Are base substitution and frameshift mutagenesis pathways interrelated? An analysis based upon studies of the frequencies and specificities of mutations induced by the (+)-anti diol

Mutation Research 326 (1995) 29-37

Fundamental and Molecular Mechanisms of Mutagenesis

Are base substitution and frameshift mutagenesis pathways interrelated? An analysis based upon studies of the frequencies

and specificities of mutations induced by the ( + )-anti diol epoxide of benzo[ alpyrene

Henry Rodriguez, Edward L. Loechler *

Department of Biology, Boston University, Boston, MA 02215, USA

Received 1 March 1994; revision received 2 August 1994; accepted 15 August 1994

Abstract

( + )-anti-B[a]PDE-induced mutagenesis is being investigated, including in a supF gene of the E. coli plasmid pUB3. Based upon various findings a working hypothesis was proposed that the major adduct of ( + )-anti-B[a]PDE (formed at N2-Gus) is able to induce different base substitution mutations (e.g., GC + TA vs. GC + AT vs. GC --f CG) depending upon its conformation in DNA, which can be influenced by various factors, such as DNA sequence context. Frameshift mutations are also significant and are analyzed herein. In virtually all cases one of three possibilities is observed: (1) some treatments change frameshift and base substitution mutation frequency (MF) in a quantitatively parallel fashion; (2) other treatments, which change frameshift MF, can change base substitution MF in a quantitatively reciprocal fashion; finally, (3) there are treatments that do not change frameshift MF, and also do not change base substitution MF. (Changes can be brought about by SOS induction, differing DNA sequence context, or heating adducted pUB3 prior to transformation. Why different kinds of changes result in (1) vs. (2) vs. (3) is discussed.) Thus, base substitution and frameshift mutagenesis pathways appear to be coupled in some way, which is most easily rationalized if both pathways are interrelated. The simplest mechanism to rationalize this coupling is that a single (+)-anti-B[a]PDE adduct in a single conformation can be bypassed via either a frameshift or a base substitution pathway. The surprising implication is that - although different conformations are likely to be required to induce different base substitution mutations (e.g., GC + TA vs. GC + AT, see above) - a single conformation can give rise to either a base substitution or a frameshift mutation. Frameshift and base substitution pathways must eventually diverge, and it is proposed that this is controlled by factors such as DNA sequence context.

Keywords: Base substitution; Frameshift metagenesis; DNA sequence context

Abbreviations: B[a]P, benzo[a]pyrene; (+ )-anti-B[a]PDE, (+ )-r-7,t-8-dihydroxy-t-9,lO-epoxy-7,8,9,1O-tetrahydrobenzo [alpyrene (anti); (+ )-anti-B[a]P-N2-Gua, the major adduct of (+ )-anti-B[a]PDE, which links the latter at its Cl0 position to Gua at the NZ position; (+ )-anti-B[a]PDE-pUB3, pUB3 adducted with _ 22 adducts of (+ )-anti-B[a]PDE/

plasmid (references 7 and 8); heated-( + )-anti-B[a]PDE, ( + )- anti-B[a]PDE-pUB3 heated for 10 min at 80°C (pH 6.8); MF, mutation frequency; D, cis-syn thymine dimer; 2-AF, 2- aminofluorene; 2-AAF, 2-acetylaminofluorene.

* Corresponding author.

0027-5107/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0027-5107(95)00149-9

1. Introduction

We have been studying mutagenesis by benzo- [alpyrene (B[a]P) (Benasutti et al., 1988; Loech- ler, 1989; Loechler et al., 1990; Mackay et al., 1992; Rodriguez et al., 1992; Rodriguez and Loechler, 1993a,b; Drouin and Loechler, 1993; Loechler, 19941, as well as dibenz[ a,j]anthracene (Gill et al., 1993a,b), which are both polycyclic aromatic hydrocarbons that belong to the family of bulky mutagens/carcinogens. B[a]P may be metabolized by a variety of pathways (e.g., Phillips et al., 1985; Marnett, 1987; Cavalieri et al., 1990; Devanesan et al., 19921, but its corresponding ( + )-anti diol epoxide (( + I-anti-B[a]PDE; Fig. 1) appears to be one important carcinogenic metabolite in many cases (Conney, 1982; Phillips, 1983; Singer and Grunberger, 1983; Harvey, 1991), where the major DNA adduct is (+)-anti- BP-N2-Gua (Cheng et al., 1989; Sayer et al., 1991).

1993). In addition, it does not appear that a significant role has to be invoked for adducts other than ( + )-anti-Bla]P-N 2-Gus to explain this base substitution mutagenic complexity (Drouin and Loechler, submitted; Jelinsky and Loechler, unpublished results). This led us to propose a potentially unifying working hypothesis (Model 1 in Rodriguez and Loechler, 1993b): (i) a single adduct (i.e., (+ )-anti-B[a]P-N2-Gua) can adopt multiple conformations; (ii) different kinds of mutations are induced by these different conformations; and (iii) adduct conformation can be controlled by various factors, such as DNA sequence context.

We have extensively analyzed base substitution mutagenesis, which primarily involves mutation at G:C base pairs where (+)-anti-B[a]PDE induces a significant fraction of GC + TA (57%), GC --f AT (23%) and GC --+ CG (20%) (Rodriguez and Loechler, 1993a,b). This complexity appeared to be composed of a minimum of two patterns: (1) principally G --f T mutations, which were observed in 5’-TG-3’ sequence contexts, as well as at the major base substitution hotspot G,,, under certain circumstances; and (2) a mixture of G -+ T, A and C mutations, which were observed in other sequence contexts, notably S-GG-3’, as well as at G,,, under other circumstances. We have ruled out a significant role for apurinic sites in (+I- anti-B[a]PDE mutagenesis (Drouin and Loechler,

Frameshift mutagenesis, which was not analyzed previously and accounts for - 34% and - 23% of mutants without and with SOS induction, respectively (Rodriguez and Loechler, 1993a,b), is considered herein. The simplest in- terpretation of the trends in the data is that the same ( + )-anti-B[alPDE adduct in the same conformation can follow either a frameshift or base substitution mutagenesis pathway.

2. Materials and methods

All materials and methods are described in Rodriguez et al. (1992) and Rodriguez and Loechler (1993a,b). In brief our system is as follows. The plasmid pUB3 has: (i) ColEl and Ml3 origins of replication; (ii) a blu gene; (iii) a 1ucZa fragment; and (iv) a supF gene, which is the mutational target. pUB3 was reacted with (+ )- anti-B[a]PDE in vitro and then either immediately transformed into E. coli ES87 cells (Apro- luc, strA/F’[pro+, IucQ, lucam26, 1uc2AM1.51) or

Scheme 1 scheme 2

UNA polymerase DNA polymerase DNA polymorascr ancounters encounters encounters adduct adduct adduct

(or other mechanisms)

&

brunchmint

base frameshift base frameshift substitution subllti.tution

Fig. 1. Two schemes to rationalize the relationship (if any) between base substitution and frameshift pathways of mutagenesis.

30 H. Rodriguez, E.L. Loechler / Mutation Research 326 (1995) 29-37

H. Rodrigzwz, E.L. Loechler/Mutation Research 326 (1995) 29-37 31

heated at 80°C for 10 min prior to transformation. When ES87 cells carry pUB3 with a wild type copy of supF, the lacZ”“26 mutation is sup- pressed, the lactose repressor is functional, and the lactose operon is repressed (off). Further- more, supF suppression of lacZ”“26 gives an uninducible lacl’ genotype, and the cells are unable to grow on lactose minimal plates. In contrast, mutagen-induced supF mutations in pUB3 prevent suppression of lacZ”“26 leading to constitutive expression of the lactose operon, which permits ES87 cells to grow on lactose minimal plates. Spontaneous mutants collected (Ro- driguez et al., 1992): 69 (MF = 0.66 x lo-‘?

-SOS; and 73 (MF = 1.03 x lOA + SOS. (+I- anti-B[a]PDE-induced mutants collected (Rodri- guez and Loechler, 1993a,b): 82 (MF = 3.8 X

lo-‘j) -SOS; 187 (MF = 20.2 x 10-6) + SOS (- heat); 254 (MF = 10.5 X 10e6) + SOS ( + heat); 137 (MF = 10.4 x 10e6) + SOS ( + freeze/thawing; see text).

3. Results and discussion

Table 1 gives information about frameshift and base substitution mutagenesis at the five significant frameshift mutational sites, which all involve

Table 1 Comparison of base pairing and frameshift deletion/insertion mutagenesis with vs. without heating at important frameshift hotspots for (+ )-anti-B[ajPDE-pUB3 studied in SOS uninduced and induced ES87 cells

Site Total MF a Total frameshift Total frameshift Total base sub- deletion MF a insertion MF a stitutionMF a

-heat - SOS -heat + SOS + heat + SOS

%oz-%os

0.56 (12) 0.65 (6) 0.58 (14)

0.56 (12) 0.22 (2) 0.25 (6)

b b b

< 0.05 (0) 0.43 (4) 0.33 (8)


%s-%o

0.23 (5) 0.54 (5) 0.50 (12)

0.093 (2) 0.22 (2) 0.25 (6)

b b b

0.14 (3) 0.32 (3) 0.25 (6)


%z-%4

0.37 (8) 2.05 (19) 0.87 (21)

0.32 (7) 1.40 (13) 0.62 (15)

b b b

0.046 (1) 0.65 (6) 0.25 (6)


%z-‘%

0.046 (1) 2.16 (20) 0.87 (21)

< 0.046 (0) 0.32 (3) 0.083 (2)

b b b

0.046 (1) 1.84 (17) 0.79 (19)


%Y%, -heat - SOS -heat + SOS + heat + SOS

%-%I,

0.37 (8) 2.59 (24) 2.40 (58)

0.23 (5) 1.62 (15) 1.41 (34)

0.28 (6) 1.30 (12) 1.32 (32)

< 0.05 (0) < 0.11 (0)

0.041 (1)

0.046 (1) 0.97 (9) 1.03 (25)

b b b

0.046 (1) 0.32 (3) 0.041 (1)

0.23 (5) 1.62 (15) 1.36 (33)

-heat - SOS < 0.05 (0) < 0.05 (0) b < 0.05 (0) -heat + SOS 0.65 (6) < 0.11 (0) b 0.65 (6) + heat + SOS 0.083 (2) < 0.04 (0) b 0.083 (2)

a MF ( x 10e6) based upon the results in Rodriguez and J_oechler (1993a,b). Values in parentheses are the total number of mutants on which MF is baaed. b No frameshift insertion mutants were detected in these cases, and the limit is < 0.05, < 0.11 and < 0.04 for -heat - SOS, -heat + SOS, and + heat + SOS, respectively.

32 H. Rodriguez, E.L. Loechler/Mutation Research 326 (1995) 29-37

deletion or insertion of a single G:C base pair in a run of G:C base pairs, as well as several other relevant sites. In Rodriguez and Loechler (1993bI, pUB3 was adducted with (+)-anti-B[a]PDE and either: (1) transformed immediately into E. coli; or (2) heated at 80°C for 10 min prior to transformation. Heating was done to probe mechanism and is also considered herein.

The relationship between the pathways for frameshift and base substitution mutagenesis can be considered based on two extreme models (schemes 1 and 2). By scheme 1, both pathways are initially related, although as indicated they must certainly diverge at some point. By scheme 2, frameshift and base substitution pathways are completely independent. In principle the frameshift pathway in scheme 2 could either involve a DNA polymerase or not (e.g., recombination).

Frameshift and base substitution mutagenesis can be reciprocally related

If scheme 1 is correct, then one would expect that in certain cases there could be a change that enhanced base substitution mutagenesis at the expense of frameshift mutagenesis (or vice versa). Three examples of this are apparent in our data set. While each of these can also be accommodated by scheme 2, it would merely be fortuitous.

Frameshift mutagenesis in runs of G:C base pairs. In our previous work, we determined that hotspots for mutagenesis were observed in most S-GG-3’ sequence contexts (Rodriguez and Loechler, 1993a). Values for [MF/S’-GG-3’1 were generally high (G,,,-G,, is the exception) and similar (0.65-1.62 x 10p6) for + SOS, as compared to MF = 0.14 X low6 for non-hotspots. Ex- cluding Gg9-G,a0, which is an unusual site (see below), Table 1 shows a trend with: base substitution mutagenesis in isolated S-GG-3’ sequence contexts (e.g., G,,,- G,,, and Gil,-Giis); a more even distribution of frameshift and base substitution mutagenesis in runs with three G:C base pairs (e.g., G,,,- G,,, and G122-G124); and a pre- ponderance of frameshift mutations in the run of five G:C base pairs (e.g., G,,,-G,,,).

It 1s from the perspective of G,72- G176 that we address this issue by asking the question: if base substitution and frameshift mutations are unre-

lated as implied in scheme 2, then why are there not more base substitution mutants obtained at G,,,-G,,,? If runs of G:C base pairs merely improve the ability of an adduct to be processed into a frameshift mutation by a slippage type mechanism (see Lambert et al., 1992 and references therein), then the base substitution mutagenesis pathway should not be affected. However, base substitution MF at G172-G176 is low, sug- gesting that frameshifts have increased at the expense of base substitutions, and that scheme 2 is less likely. It is as if DNA polymerase is encountering the same problem in all of these 5’- GG-3’ sequences, but is solving the problem differently (e.g., into frameshifts at G,,*-G,,, and into base substitutions at G1i5-G1J as dictated by the DNA sequence context surrounding the adduct. This argues for an interrelationship between frameshift and base substitution mutagenesis as implied in scheme 1.

G,,,-G,,, and G,2r-G,,, frameshift mutations. Nineteen mutations were detected in the run of three G:C base pairs at G,,s-G,,, ( + SOS); 13 were frameshift mutations and six were base substitution mutations (Rodriguez and Loechler, 1993a). Although a similar number of total mutations (i.e., 20) were detected in the run of three G:C base pairs at G,,,- G,,, (+ SOS), there were fewer frameshift mutations, three, but more base substitution mutations, 17.

In this case it is as if the same number of adducts at G,Os-G,,, vs. G,,,-G,, were processed into the same number of mutations as expected if mutational hotspots are defined by 5’-GG-3’ sequence contexts. It is as if DNA polymerase encounters the same problem at both runs, but solves the problem differently depending upon some factor, such as long-range sequence context that extends beyond the G:C base pairs. These results are most easily rationalized by scheme 1, especially given some of the results involving heating described below.

G,,-G,,, frameshijt mutations. Frameshift MF ( = 0.22 x lo-? + SOS appears to be smaller than -SOS (MF = 0.56 x lO-‘j). Although this decrease is not statistically significant given that frameshift MF + SOS is based on only two mutants, it is likely to be real for the following

H. Rodriguez, E. L. Loechler /Mutation Research 326 (1995) 29-37 33

reasons. A similar MF (= 0.25 X lo-‘? was obtained + SOS/ + heating based upon six mutants collected (Table l), and - in data not discussed extensively herein - if the heating step is re- placed by freeze/thawing, MF (= 0.23 X 10p6) is also similar based upon three mutants collected (Rodriguez and Loechler, 1993b). Thus, three sets of data (i.e., unheated, heated and freeze/ thawed) gave a frameshift MF + SOS of 0.22- 0.25 X 10m6, which suggests that the true value is likely to be reasonably close to this range, and that SOS induction does indeed decrease frameshift MF by N 2.5fold at G,,-G,,.

It is likely that deletions at G,,-G,, must follow a different pathway than deletions at other sites both because of an SOS-induced decrease in frameshift mutagenesis, which is atypical in our data set and unprecedented to our knowledge, and because this is the only site where frameshift MF is significant with only two consecutive G:C base pairs. The sequence around G,,-Gioo might promote a mechanism of frameshift mutation (Fig. 2) based upon template-mediated events (re- viewed in: Ripley, 1990; Kunkel, 1990, 1992).

For the purposes of illustration, consider an adduct at G,, which might block replication (Fig. 2: steps 1). ’ In step 2, primer slippage would permit dATP to be incorporated into the growing primer strand (step 3). In step 4 the primer would reanneal to the template beyond the (+)-anti- B[a]PDE adduct, which would be looped out in the template strand. Replication would then con- tinue (step 5).

In contrast to frameshift mutagenesis, SOS induction increased base substitution MF > N 8- fold at G,,-G,, (from < N 0.05 X 10e6 to _ 0.43 x 1O-6). In fact base substitution plus frameshift MF at G,,-G,, (- 0.65 x lo-‘? + SOS is reasonably similar to MF for frameshift mutagenesis at G,,-G,, (N 0.56 x lo-? - SOS. It is as if SOS induction has not affected the fraction of adducts processed into mutations, but has instead shifted the pathway from one domi-

‘Anduct at Gloo might also be responsible for G,-G,, frameshift mutations, although G, seems more likely given this is the hotspot for base substitution mutations.

Jo 1

CACCCC-5 ’ 5’-CTGTGGTGGGG-3’

C-ACCCC-5 ’ 5 ’ -CTG TGGGG-3 ’

TG w

I

BP50 .

AC%CCC-5 ’ 5’-CTG TGGGG-3’

TG w

I BP

Jo 4

A*CACCCC- 5 ’ 5 ’ -CTGT GTGGGG-3 ’

\G’

I

BP J@ (9 4iCA>A&CC- 5 ’ 5’-CTGT GTGGGG-3’

\G’

I BP

Fig. 2. A model to rationalize why G,-GIoo might be a hotspot for frameshift mutagenesis assuming an adduct at G, (see footnote 1).

nated by frameshift mutagenesis -SOS to a pathway where base substitution mutagenesis is more prevalent + SOS. This suggests that DNA polymerase is encountering the same problem with an adduct either -SOS or + SOS induction, but is solving the problem differently following SOS induction. This argues for scheme 1. Pre- sumably, this pattern is only observed at G,,-G,, because frameshift pathways at other sites involve a different mechanism, which can be enhanced by SOS induction (discussed in more detail below).

Frameshift and base substitution mutagenesis can change in parallel

If frameshift and base substitution pathways are interrelated (scheme 11, then there may also be examples where treatments increase (or decrease) base substitution and frameshift mutagenesis in parallel. (Below we discuss why it is sensi-

34 H. Rodriguez, E.L. Loechler /Mutation Research 326 (199.5) 29-37

ble that parallel changes might occur in certain cases, while reciprocal changes occur in others.) At both G,,,-G,,, and G,,,-Gi,, heating led to a decrease in both base substitution and frameshift mutagenesis (Table 1). Furthermore, the decrease is remarkably similar, - 2.4-fold, in all cases but one, where it is N 3.9-fold (frameshift mutagenesis at G,,,- GiZ4), although the latter is not statistically significantly different. By whatever mechanism the fact that heating affects base substitution and frameshift mutagenesis virtually identically at both G,as-G,,, and G,,,-G,,, is more easily rationalized by scheme 1 than scheme 2. Considering Gg9-G1m, heating of (+)-anti- B[alPDE-pUB3 did not affect frameshifts; thus, if scheme 1 were correct, then base substitutions should also be unaffected, which appears to be the case (Table 1).

Only one piece of data may argue against scheme 1. G 172-G,76 is the one site where there does not appear to be a correlation between the behavior of frameshift mutagenesis, which is not affected by heating, and base substitution mutagenesis, which appears to decrease upon heating (Table 1). However, frameshift mutagenesis over- whelmingly dominates at G172-G176, and, in fact, the perceived decrease in base substitution MF is based upon a small number of mutants (3 vs. 1 for + heat vs. -heat, respectively). Although these results should not be dismissed, it seems unreasonable to rule out scheme 1 based upon them.

G,,,-G,,, is relatively refractory to mutagenesis based upon a comparison to runs with fewer (i.e., G,,,-G,,, and G122-G124) and more (i.e., G,,,-G,,,) G:C base pairs. There are no striking trends to analyze, although these results are easily accommodated by scheme 1.

Are frameshift and base substitution mutagenesis always coupled?

In most cases described above, the data fit into one of three categories: (1) a change in frameshift mutations was associated with a quantitatively reciprocal change in base substitutions; (2) a change in frameshift mutagenesis was associated with a quantitatively parallel change in base substitutions; (3) no change in frameshifts was associ-

ated with no change in base substitutions. This coupling tends to argue that base substitution and frameshift mutagenesis may indeed be interrelated and that scheme 1 is more likely than scheme 2.

It is important to discuss one kind of comparison where coupling is not evident. When MF for -SOS vs. + SOS is compared for all sites but

G99-G1007 which is unusual as discussed above, although frameshift and base substitution mutagenesis increase in parallel, the magnitude of the increase frequently appears quite different (Table 1). This trend could either be an argument in favor of scheme 2, or might suggest that frameshift and base substitution mutagenesis pathways are not completely coupled. In this regard, two points are considered.

(1) In all cases, comparisons between MF for -SOS vs. + SOS cannot be made reliably because an insufficient number of mutants were collected. Nevertheless, these differences in SOS induction cannot be completely discounted: e.g., the exact SOS enhancement of frameshift insertion MF at G,,,- G,,, is uncertain, but it is certainly much larger than for base substitutions.

(2) Scheme 1 only implies that the initial phase of frameshift and base substitution mutagenesis pathways is similar; thereafter, the pathways must certainly diverge. It seems reasonable to imagine that SOS induction affects mutagenesis only after the pathways diverge, in which case it could have a differential effect on the frameshift vs. the base substitution pathway. This is certainly consistent with the notion that the SOS gene products inter- act with the editing apparatus of DNA polymerase III (see Slater and Maurer, 1991 and references therein).

How could base substitution and frameshift mutagenesis pathways be related?

If scheme 1 is correct, it implies that the same adduct is responsible for inducing both base substitution and frameshift mutations. In the case of ( + )-anti-B[a]PDE, the likely candidate is the major adduct ( + )-anti-B[a]P-N *-Gua, although this has not been demonstrated in a site-specific study. Of course, it is unknown which adducted Gua in a run is responsible for a frameshift mutation.

H. Rodriguez, E.L. Loechler /Mutation Research 326 (1995) 29-37 35

However, if adducts in different positions were responsible for the frameshift vs. the base substitution mutations in a run, then one would not expect to see such a seemingly simple relationship between base substitution and frameshift mutagenesis.

As noted above GC + TA, GC + AT and GC + CG (ratio 57:23:20) are all prevalent. Our working hypothesis to rationalize this complexity is that (+ l-anti-B[a]P-N*-Gua can adopt different conformations leading to different mutations, and we have proposed that there are at least two mutagenic conformations (Rodriguez and Loech- ler, 1993b). If we accept for the moment that adduct conformational complexity is at the root of mutational complexity, then: are the conformations of the adduct yielding frameshift mutations the same as those yielding base substitution mutations or not? What follows would argue that they are.

Consider G,,,-G,,a and G122-G124, where heating decreased MF for both base substitution and frameshift MF similarly. These results are most easily rationalized if both frameshift and base substitution mutations occur from one conformation of the adduct, but where there is an- other conformation from which mutations principally do not occur. (In this regard, > 90% of the time bulky adducts studied site-specifically, including ( + l-anti-B[a]P-N *-Gua, do not induce a mutation (discussed in Loechler, 1994j.j If the mutagenic and non-mutagenic conformations are not in rapid equilibrium, then heating could induce a net conversion from the more to the less mutagenic conformation, which would lead to a net decrease m MF at G,,,- G,,, and G122-(3124

following heating. 2 At other sites (e.g., G,-G,, and G,,,- G,,,), the mutagenic and non-mutagenic conformations may be in equilibrium, such that heating has no effect on mutagenesis.

If true, this would imply at least three conformations: two leading to different hinds of muta-

‘Heating might also lead to the loss of adducts at G,,-

G tra and G~zz-GIB, which could decrease MF by several

mechanisms. Various results make this possibility less likely

(Rodriguez and Loechler, 1993b; Drouin and Loechler, 1993).

tions and one not giving mutations. Scheme 1 can only apply if both frameshift and base substitution mutational pathways begin from the same initial conformation of a DNA adduct. 3 This suggests that base substitution and frameshift pathways are more interrelated than different types of base substitution pathways, which appear to require different conformations. For example, we have evidence that heating can convert G + T mutations into G + A and G --) C mutations (Rodriguez and Loechler, 1993b), but there is no evidence that heating can convert a base substitution mutation into a frameshift mutation or vice versa.

Based on the premise that adduct conformational complexity is at the root of mutational complexity, the data herein would be rationalized as follows. Adduct structure is affected by heating prior to transformation and, thus, before encountering a DNA polymerase, would be expected to affect both frameshift and base substitution mutagenesis in parallel based on scheme 1 as observed (e.g., Gras- G,,, and G,,-G,&. Adduct structure might be very similar in S-GG-3’ sequences, but partitioning toward the base substitution vs. frameshift pathways could be influenced by longer range DNA sequence context effects after the initial encounter, such that frameshift mutations dominate in longer runs of G:C base pairs (e.g., G,,,-G,,J, while base substitution mutations dominate in isolated S-GG-3’ sequence contexts (e.g., G,rS-G,,,). It seems most likely that SOS induction has an effect after the divergence point in scheme 1, so base substitution and frameshift pathways would not necessarily be expected to be affected by SOS induction similarly (e.g., G,,,- Gi,J. If both base substitution and frameshift mutations are enhanced by SOS induction, it implies that less bypass occurs from the mutagenic conformation -SOS and the

3 Each of these three conformations is proposed to be

relatively different from each other, such that - at least in

certain cases - they do not readily interconvert unless heated.

It is important to note that each of these unrelated conforma-

tions could be composed of several closely related conforma-

tions that can rapidly interconvert.

36 H. Rodriguez: E. L. Loechler / Mutation Research 326 (1995) 29-37

adduct may block replication and potentially be lethal. On the other hand if SOS induction does not affect overall MF, but enhances base substitution compared to frameshift mutagenesis (e.g., as appears to be the case at G,,-G,,), then base substitution mutagenesis will increase at the expense of frameshift mutagenesis according to scheme 1 as observed.

Finally, it is important to note that there is some evidence for the presence of multiple conformations of the major tram addition adduct of ( + )-anti-B[a]P-N2-Gua (discussed in Loechler, 19941, although no structures are known. How- ever, it is well-established that different enan- tiomeric adducts of anti-B[a]P-N2-Gua can adopt at least three different conformations (Cosman et al., 1992, 1993; de 10s Santos et al., 19921.

b there evidence that base substitution and frameshift mutagenesis are interrelated for other mutagens?

Wang and Taylor (1992) have studied a ci.s-syn thymine dimer at all five positions in a run of six consecutive thymine residues in vitro. Frameshift mutations only occurred at a significant frequency (N 30% and -5% for -1 and -2 frameshifts, respectively) in the case of one construct: 5’-TDTIT-J, where D represents the thymine dimer. The result was that this was also the only construct that gave rise to base substitution mutations at a significant frequency ( N 30%). This result strongly suggests that there is some- thing unique about the thymine dimer in the S-TDTTT-3’ context (perhaps conformation), which promotes both base substitution and frameshift mutagenesis, in comparison to the other four contexts. This may simply be the pref- erential ability of DNA to slip when the thymine dimer is in this position, which may suggest that slippage promotes base substitution, as well as frameshift mutagenesis. By whatever connection, this finding seems to support the notion that frameshift and base substitution mutagenesis pathways can be interrelated.

The in vitro results of Wang and Taylor (1992) clearly show that frameshift deletion mutagenesis involving DNA lesions can occur during a polymerization step. Scheme 1 also implies that

frameshift mutations are initiated during DNA polymerization with ( + )-anti-B[a]PDE, and argues against the significance of other mechanisms, such as those involving recombination.

Acknowledgements

We thank Elise Drouin, Rasheed Khalid and Scott Jelinsky for helpful discussions. We grate- fully acknowledge the Cancer Research Program of the National Institute, Division of Cancer Cause and Prevention, Bethesda, MD, for provid- ing ( + )-anti-B[a]PDE. This work was supported by NIH Grant ES03775 and ACS Grant CN-54.

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Documents

Are base substitution and frameshift mutagenesis pathways interrelated? An analysis based upon studies of the frequencies and specificities of mutations induced by the (+)-anti diol