A hypothesis for what conformation of the major adduct of (+)-anti-B[a]PDE (N2-dG) causes G->T versus G->A mutations based upon a correlation between mutagenesis and molecular modeling results

Richard E. Kozack, Rajiv Shukla and Edward L. Loechler1

Department of Biology, Boston University, 2 Cummington Street, Boston, MA 02215, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Molecular modeling (simulated annealing) was used to study the conformations in dsDNA of [+ta]-B[a]P–N2-dG (R.E.Kozack and E.L.Loechler, accompanying paper), which is the major benzo[a]pyrene (B[a]P) adduct. Sixteen classes of conformations were identified, and are analyzed herein vis-a-vis the two most prominent B[a]P mutations, G->T and G->A base substitutions. Eight conformations seem more relevant to frameshift mutagenesis, so they are excluded, leaving eight conformations as follows. Two conformations (BPmi5 and BPmi3) retain Watson–Crick G:C base pairing having the B[a]P moiety of the adduct in the minor groove. Two conformations (BPma5 and BPma3) have the Hoogsteen orientation with B[a]P in the major groove. Four conformations are base displaced and have B[a]P stacked in the helix with the dG moiety of the adduct displaced into either the major groove (Gma5 and Gma3) or the minor groove (Gmi5 and Gmi3). Three of these eight conformations (BPma5, BPma3 and Gma3) are universally high in energy. The two conformations that retain G:C base pairing potential (BPmi5 and BPmi3) are likely to be non-mutagenic. Of the three remaining conformations, Gmi5 can be relatively low in energy, but is distorted. A correlation exists between the calculated energies for the remaining two base displaced conformations and mutagenesis for [+ta]-B[a]P–N2-dG, leading to the hypothesis that Gma5 is responsible for G->T mutations and Gmi3 is responsible for G->A mutations. Gma5 and Gmi3 resemble each other, except that dG is in the major and minor grooves, respectively. An incipient rationale for this hypothesis is discussed: DNA polymerase might be triggered to follow a different mutagenic pathway depending upon whether a non-informational lesion has bulk protruding into the major or minor groove. A pathway for interconversion between these eight conformations is also proposed and its implications are discussed; e.g. four steps are required to interconvert between Gma5 and Gmi3.

Abbreviations: (+)-anti-B[a]PDE, (+)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10,-tetrahydrobenzo[a]pyrene (anti); B[a]P, benzo[a]pyrene; [+ta]-B[a]P–N2-dG, the major adduct of (+)-anti-B[a]PDE formed by trans addition of N2-dG to (+)-anti-B[a]PDE; ds, double-stranded; ss, single-stranded.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The potent mutagen and carcinogen benzo[a]pyrene (B[a]P) is metabolized to its carcinogenic form, (+)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti) [(+)-anti-B[a]PDE] (Figure 1Go), which reacts with DNA at N2-dG in duplex DNA and gives a major adduct, [+ta]-B[a]P–N2-dG) (19). We have been studying both the mutations induced by and the conformations adopted by [+ta]-B[a]P–N2-dG in order to understand which conformations are most likely to be responsible for which mutations and why (1020). [This subject is introduced more thoroughly in an accompanying paper (21).]



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Fig. 1. The eight conformations of [+ta]-B[a]P–N2-dG most likely to be relevant to base substitution mutagenesis (see text) and a hypothetical scheme for how they might interconvert. [More information about this type of presentation can be found in the legend to Figure 1Go in the accompanying paper (21).] The B[a]P moiety of [+ta]-B[a]P–N2-dG is depicted as a half open and half solid rectangle with the solid portion representing the a-face, which is the face commonly viewed when B[a]P is drawn. The dG moiety of the adduct is a rectangle with a G towards its center. The base pair on the 3'-side of the adduct is always located toward the top of the adduct and the base pair on the 5'-side of the adduct is always located toward the bottom of the adduct. The major groove is always oriented to the left and the minor groove to the right of the adduct. The abbreviations that represent each of the conformations are based on the designation of the moiety in the groove. For example, BPma5 indicates that the B[a]P moiety of the adduct is in the major groove and pointing toward the base on the 5'-side of the adduct. Gmi5 indicates that the dG moiety is in the minor groove; however, in this case, the 5 indicates that the a-face of the B[a]P moiety of the adduct is pointing toward the base on the 5'-side, which is equivalent to saying that the adduct bond (directionality C10 of B[a]P->N2-dG) is pointing toward the base on the 5'-side. Each base paired conformation is related to a corresponding base displaced conformation by an S-type transition, which involves a replacement in the helix stack of the dG moiety of the adduct by the B[a]P moiety of the adduct by a simple sliding motion (Figure 5Go). In addition, when the B[a]P moiety is in the major or minor groove, it can rotate (R) about the adduct bond (Figure 5Go). These six transitions lead to the grouping of the eight conformations into two sets of four conformations each. The most logical point of transition between these two sets is via BPmi3{leftrightarrow}Gmi3, which is indicated by dashed arrows and probably involves two steps (see text).

 
[+ta]-B[a]P–N2-dG appears to be able to adopt at least 16 classes of conformations in double-stranded (ds)DNA (21). This is a large number of conformations, so we have used a combination of reasoning and molecular modeling to exclude some of these in an effort to try to generate a reasonable and testable hypothesis for what conformation might be responsible for G->T versus G->A mutations induced by [+ta]-B[a]P–N2-dG. Eight of the 16 conformations seem more likely to be relevant to frameshift mutagenesis because they have either one fewer or one more step (21) in the DNA strand containing the adduct than in the complementary strand.

In contrast, the remaining eight conformations have the same number of steps in both DNA strands and seem more likely to be relevant to base substitution mutagenesis. These eight are shown in Figure 1Go, which illustrates the conformations pictorially and includes the abbreviations that we have adopted to represent each conformation. (The organization of the conformations in Figure 1Go is discussed in Results and discussion). Parts of each of these eight conformations, including the B[a]P and dG moieties of the adduct, are shown in stereo pairs in Figures 2 and 3GoGo. The orientation of the images in Figures 1–3GoGoGo is as follows: the base on the 5'-side of the adduct is down; the base on the 3'-side of the adduct is up; the major groove is left; the minor groove is right.



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Fig. 2. Stereo pairs of the conformations Gma5, Gma3, BPmi5 and BPmi3. [+ta]-B[a]P–N2-dG and its complementary dC are shown toward the center of each panel with the base pairs on the 5'- and 3'-sides of the adduct being below and above, respectively. The major and minor grooves are to the left and right, respectively. The sequence context is 5'-TGC-3'.

 


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Fig. 3. Stereo pairs of the conformations Gmi5, Gmi3, BPma5 and BPma3. [+ta]-B[a]P–N2-dG and its complementary dC are shown toward the center of each panel with the base pairs on the 5'- and 3'-sides of the adduct being below and above, respectively. The major and minor grooves are to the left and right, respectively. The sequence context is 5'-TGC-3'.

 
In two of the eight conformations (BPmi5 and BPmi3), the dG moiety retains the ability to form a Watson–Crick base pair with its complementary dC and the B[a]P moiety is in the minor groove pointing toward the base on either the 5'- or the 3'-side, respectively. Two other conformations (BPma5 and BPma3) result when the dG moiety of the adduct undergoes an anti->syn base rotation about the glycosylic bond to give a Hoogsteen G:C base pair; this places the B[a]P moiety in the major groove and pointing toward the base on the 5'- or 3'-side, respectively. Two conformations (Gma5 and Gma3) are base displaced, in that the guanine moiety of the adduct is displaced into the major groove and is replaced in the helix by the B[a]P moiety. Each is stacked with an opposite orientation of the planar B[a]P moiety, where the `a-face' is pointing toward the 5'- or 3'-side, respectively. The a-face is defined in Figure 1Go. [This is equivalent to saying that the adduct bond is pointing toward either the base on the 5'- or the 3'-side, respectively (Figure 1Go) (see also ref. 21).] Finally, two of these conformations (Gma5 and Gma3) are base displaced and have the B[a]P moiety stacked in the helix with the guanine moiety of the adduct in the minor groove with the a-face (and the adduct bond) of the B[a]P moiety either pointing toward the base on the 5'- or the 3'-side, respectively.

It is important to emphasize that the intent of our work was to generate a hypothesis based on what appears to us to be logical, in order to provide a starting point for future theoretical and experimental studies. Our work assumes that information about the conformations of [+ta]-B[a]P–N2-dG opposite dC in dsDNA has some relevance to mutagenesis, which actually involves the conformation of a transition state during the incorporation of any base other than dC at a single-stranded (ss)DNA–dsDNA junction in the presence of a DNA polymerase (i.e. kb1 and kb2 in Figure 4Go). Certainly, the `mutagenic mechanism point' must occur at a ssDNA–dsDNA junction in the presence of DNA polymerase; however, if interconversion between conformations in this state is slow (i.e. k2, k–2 < kb1, kb2 in Figure 4Go), then the pathway of mutagenesis is dictated (i.e. the `decision point') by the mixture of conformations established in duplex DNA. This issue has not been settled definitively, but we believe that interconversion between the conformations responsible for G->T versus G->A mutations is likely to be relatively slow (10,13,14,16,17), so we have modeled [+ta]-B[a]P–N2-dG in dsDNA, which is our best guess as to where the `decision point' is. Some of our data (principally ref. 13, but also refs 10,14,16,17) can best be rationalized if k1 and k–1 (Figure 4Go) are slow at 0°C and k2 and k–2 are slow at 37°C. Equilibration via k1 and k–1 occurs upon heating at 80°C for 10min or by freeze/thawing. (Alternatively, trends for k1/k–1 versus k2/k–2 may be similar, which would also make modeling in dsDNA indirectly revealing about mutagenesis.)



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Fig. 4. Rationale for why we think [+ta]-B[a]P–N2-dG in dsDNA is relevant to the pathway of mutagenesis. The G->T mutagenic pathway is in the top row (ka1 and kb1) and involves incorporation of dATP opposite one conformation of [+ta]-B[a]P–N2-dG (BP). The G->A mutagenic pathway is in the bottom row (ka2 and kb2) and involves incorporation of dTTP opposite a second conformation of [+ta]-B[a]P–N2-dG (BP). The `mutagenic mechanism point' is the place where dATP or dTTP is incorporated (kb1 and kb2) and occurs at a ssDNA–dsDNA junction in the presence of DNA polymerase. However, if k2 and k–2 are relatively slow with respect to kb1 and kb2, then the `decision point`, which dictates the pathway to be followed, is actually in dsDNA and the fraction of G->T versus G->A mutations is determined by the ratio of conformations associated with k1 and k–1.

 
Herein, we discuss why we believe that the eight conformations most germane to base substitution mutagenesis can be grouped into two sets of four conformations each, based upon their ability to interconvert via motions that would be relatively non-disruptive to DNA structure (Figure 1Go). We also discuss the most likely point where these two groups of four conformations might interconvert. Finally, we analyze the eight conformations and suggest a hypothesis that Gma5 and Gmi3 are responsible for G->T and G->A mutations, respectively. Gma5 and Gmi3 have a remarkably similar profile in dsDNA, except that the dG moiety is in the major and minor grooves, respectively, which suggests a possible rationale for their mutagenic differences.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All methods are described by Kozack and Loechler (20,21).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In the accompanying paper (21) we argue that eight of 16 possible conformations are most likely to be potentially relevant to base substitution mutagenesis (Figure 1Go). (These conformations are described in the Introduction and the legend to Figure 1Go.) A canonical version of each of these eight conformations was constructed by docking [+ta]-B[a]P–N2-dG in dsDNA and was refined using a combination of conjugate gradient minimization and a molecular dynamics version of a simulated annealing protocol (21). These eight conformations were evaluated in each of five different DNA sequence contexts (Table IGo). Twenty-five simulated annealing runs were conducted on each of these 40 conformations. Each simulated annealing run differed by systematically varying the annealing parameters based on our previous work (20,21). Conclusions regarding adduct conformations and energies are discussed in the accompanying paper (21) and herein we consider interconversion between these eight conformations, as well as mutagenesis.


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Table I. Comparisons of the relative energies for the conformations Gma5, Gmi3 and Gmi5 with the kinds of base substitution induced by [+ta]-B[a]P-N2-dG in five sequence contexts
 
Conformational interconversions
Figure 5Go illustrates one conformational interconversion that is relatively non-disruptive to DNA structure. The base displaced Gma5 conformation could interconvert to the base paired BPmi5 conformation by a simple motion with the B[a]P moiety moving from being base stacked to being in the minor groove and with the dG moiety of the adduct moving from the major groove into the helix, where it becomes base paired with its complementary cytosine. We refer to this as a `slide' (S). Each of the four base paired conformations is related to a single base displaced conformation by a S-like transition analogous to that depicted in Figure 5Go. A `rotation' (R) about the adduct bond also appears relatively non-disruptive when the B[a]P moiety is either in the major or minor groove, giving two additional interconversions. The combination of the four slides and two rotations leads logically to grouping of the eight conformations into two sets, each containing four conformations (Figure 1Go).



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Fig. 5. A pictorial representation of an S-type transition (slide) between Gma5 and BPmi5, as well as the R-type transition (rotation) between BPmi5 and BPmi3 for [+ta]-B[a]P–N2-dG. The S-type transition shows the B[a]P moiety of the adduct moving out of the helix and into the minor groove, while concurrently the dG moiety moves from the major groove into the helix. Model building shows that this transition is relatively unfettered, although it does require dsDNA to become somewhat distorted. The R-type transition is a simple rotation of the B[a]P moiety in the minor groove about the adduct bond from being pointed toward the base on the 5'-side to being pointed toward the base on the 3'-side. In contrast, a direct transition from Gma5 to BPmi3 requires a slide and a rotation to occur more or less simultaneously and there is no room for this rotation without seriously distorting the DNA.

 
Considering the 1000 molecular modeling (simulated annealing) runs that we did, one conformation interconverted to another in 13 instances. There were five examples of an S-type motion: Gma5->BPmi5 (5'-CGG-3'); BPma5->Gmi5 (5'-CGG-3' and 5'-GGG-3'); Gma3->BPmi3 (twice with 5'-CGG-3'). There were eight examples of R-type motion: BPmi3->BPmi5 (twice with 5'-CGG-3', twice with 5'-AGA-3' and thrice with 5'-GGG-3'); BPma5->BPma3 (5'-CGG-3'). In each of these cases the final conformation was relatively high in energy, although it was clear that an interconversion had occurred. Thus, of the six transitions depicted in Figure 1Go, examples of each were observed with the exception of Gmi3 {leftrightarrow}BPma3. These findings support the notion that the S-type and R-type transitions are relatively plausible.

Recently, a BPmi5->Gma5 transition was observed in another molecular modeling study (23). Indirect observation of a BPmi5->Gma5 conversion was made by NMR for [+ta]-B[a]P–N2-dG, in that BPma5 is preferred in fully duplex DNA, but at a ssDNA–dsDNA junction Gma5 is observed (24). Finally, Geacintov et al. have analyzed the conformations of various stereoisomers of B[a]P–N2-dG adducts and have noted that BPmi5 and Gma5 appear to be related, notably in that they share many of the same torsion angles (25). These observations firmly establish the sensibleness of the BPmi5 {leftrightarrow}Gma5 transition and suggest that the other S-type transitions are probably also sensible.

Figure 5Go also illustrates what would have to occur for a transition from Gma5 to BPmi3 to occur directly in a single step. Such a direct transition is expected to be very disruptive to DNA structure, since it requires the B[a]P moiety not only to move into the minor groove, but also to flip over in the process, which is hard to imagine being a low energy process because there is no room for the B[a]P moiety to flip over intrahelically. Thus, it seems more likely that interconversion between Gma5 and BPmi3 would involve an S-type motion to give BPmi5, followed by an R-type motion to give BPmi3.

With the exception of the four S-type and two R-type interconversions depicted in Figure 1Go, there are no other simple transitions between any of the other pairs of conformations. In all other cases, the process would be either multi-stepped or relatively conformationally strained, e.g. as illustrated for the Gma5->BPmi3 transition in Figure 5Go. In fact, during our studies of the 1000 conformations, there were no examples of conformational interconversions with the exception of those that we have classified as being either S-type or R-type.

The thinking in the previous paragraph leads to the question: how does the Gma5/BPmi5/BPmi3/Gma3 set of conformations interconvert with the Gmi5/BPma5/BPma3/Gmi3 set? It appears that there are four possible points of interconversion: BPmi3{leftrightarrow}Gmi3, BPmi5{leftrightarrow}Gmi5, BPma3{leftrightarrow}Gma3 and BPma5{leftrightarrow}Gma5. For each of these interconversions, two pathways are possible, each requiring two steps (Figure 6Go). By the first pathway, the dG moiety might move into the relevant groove (e.g. BPmi5->G/BPmi5), after which the B[a]P moiety can replace it in the base stack (e.g. G/BPmi5->Gmi5). Alternatively, the B[a]P moiety might first intercalate (e.g. BPmi5->Imi5) and thereafter the dG moiety of the adduct might swing from the stack into the relevant groove (e.g. Imi5->Gmi5). Model building shows that, because DNA is right-handed, the BPmi3{leftrightarrow}Gmi3 transition is less sterically hindered than the BPmi5{leftrightarrow}Gmi5 transition and the BPma3{leftrightarrow}Gma3 transition is less sterically hindered than the BPma5{leftrightarrow}Gma5 transition (data not shown). Finally, because both BPma5 and Gma5 appear to be high energy conformations (21) and have never been observed by (for example) NMR, while both BPmi3 and Gmi3 are calculated to be lower in energy and have been observed by NMR (reviewed in ref. 25), it seems that the BPmi3{leftrightarrow}Gmi3 transition is the most likely place to imagine that the two sets of four conformations might interconvert (Figure 1Go).



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Fig. 6. Possible pathways for interconversion of [+ta]-B[a]P–N2-dG between pairs of conformations relevant to base substitution mutagenesis, in each case requiring intermediates postentially relevant to frameshift mutagenesis, including conformations referred to as being double displaced (e.g. G/BPmi5) or intercalated (e.g. Imi5) [(see accompanying paper (21)].

 
Potential relationships between [+ta]-B[a]P–N2-dG conformations and mutagenesis
As discussed in the accompanying paper (21), [+ta]-B[a]P–N2-dG can adopt at least 16 classes of conformations in dsDNA. Eight of these were excluded because they were more likely to be relevant to frameshift mutagenesis, while three (BPma5, BPma3 and Gma3) were almost universally calculated to be high in energy. A variety of results suggest that the major event that occurs when [+ta]-B[a]P–N2-dG is bypassed during replication is correct incorporation of dC (11,26,27). Evidence suggests that a different conformation is associated with mutagenic versus non-mutagenic bypass (10,13,16). BPmi5 and BPmi3 are the two most likely candidates to be associated with non-mutagenic bypass, since the dG moiety of the adduct retains the potential to form a relatively normal Watson–Crick base pair.

This leaves three base displaced conformations, Gma5, Gmi5 and Gmi3, as being most likely to be relevant to mutagenesis. The notion that conformations like BPmi5 and BPmi3, which retain base pairing potential, give rise to no mutations, while base displaced conformations do cause mutations was probably first articulated by Broyde and Hingerty (28) and has been seconded in a number of cases (29), including by us (10).

One of our objectives has been to find a correlation between something in our conformational studies and our mutational studies that might point us toward a hypothesis for [+ta]-B[a]P–N2-dG mutagenesis. Table IGo shows a ranking of the relative energies of the three base displaced conformations, Gma5, Gmi3 and Gmi5, in the five sequence contexts most relevant to mutagenesis and also reports on our mutagenesis results in these same five sequence contexts based on our adduct site-specific mutagenesis studies with [+ta]-B[a]P–N2-dG (11,15,18,19,30). The energies were averaged for the three lowest energy examples for each individual conformation, as well as for the single lowest conformation (in parentheses) based on the results in Figures 2–6GoGoGoGoGo in the accompanying paper (21). The relative energies are reported using Gma5 arbitrarily as a reference. A correlation exists where Gmi3 is lower in energy when G->A mutations predominate (5'-CGT-3' and 5'-AGA-3'), Gma5 is lower in energy when G->T mutations predominate (5'-TGC-3'), while Gma5 and Gmi3 are more similar in energy when G->T and G->A mutations are more evenly distributed (5'-CGG-3' and 5'-GGG-3'). This correlation is illustrated quantitatively by plotting the fraction of [(G->T)/(G->A)] mutations (as the log) versus the relative energy of the Gma5 versus Gmi3 conformation (Figure 7Go), which gives a reasonable straight line. The energy for Gmi5 does not correlate; thus, we are less inclined to consider it in mutagenesis; it is also worth recalling that [+ta]-B[a]P–N2-dG can block replication (11,26,27) and Gmi5 could be associated with this. We are less inclined to associate Gmi5 with mutagenesis for several additional reasons. Gmi5 has not been observed to date in any NMR study of B[a]P or related adducts, while Gma5 and Gmi3 (as well as BPmi5 and BPmi3) have (reviewed in ref. 25). Gmi5 is a relatively unattractive structure, where the complementary dC overlaps the B[a]P moiety of the adduct (Figures 3 and 8GoGo), which intuitively seems less likely to be potentially involved in a replication event. In contrast, Gma5 and Gmi3 have remarkably similar conformational profiles and principally differ only in that their dG moiety is in a different groove (Figure 8Go).



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Fig. 7. A plot of the log of the fraction of [G->T]/G->A] mutations versus the difference in energy between the conformations Gma5 and Gmi3 in five DNA sequence contexts for [+ta]-B[a]P–N2-dG (data from Table IGo). The solid line is the best straight line (slope 0.143, intercept 0.70) associated with the solid circles, which are derived from values for the energies when the average of the three lowest conformations for Gma5 and Gmi3 are used. The dashed line is the best straight line (slope 0.099, intercept 0.56) associated with the open circles, which are derived from values for the single lowest energy conformation for Gma5 and Gmi3. In essence, when Gma5 is calculated to be lower in energy, then G->T mutations are preferred and when Gmi3 is calculated to be lower in energy, then G->A mutations are observed.

 


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Fig. 8. Comparison of the lowest energy versions of [+ta]-B[a]P–N2-dG in the conformations Gma5, Gmi3 and Gmi5 (as well as a normal G:C base pair) when viewed down the helix axis in a 3'->5' direction. The sequence context is 5'-TGC-3'. In the case of the Gmi5 conformation, the dC moiety is actually in a plane above the B[a]P moiety.

 
Gma5, Gmi3 and mutagenesis
The goal of our molecular modeling work was not to come to a conclusion about what conformations were responsible for [+ta]-B[a]P–N2-dG base substitution mutagenesis, but rather simply to generate a hypothesis that could be tested both theoretically and experimentally. The hypothesis that Gma5 and Gmi3 are responsible for G->T and G->A mutations, respectively, is testable and experiments are underway in this regard.

There are a number of reasons why this correlation might be inconsequential, including that it could be a fortuitous artifact of the inherent inexactness of molecular modeling calculations or that it could be that we are modeling this adduct in pure dsDNA, which might not be germane to mutagenesis (discussed below). It is our hope that the presentation of this hypothesis, even if proven wrong, will contribute to expanding discussion of the possible relationship between adduct conformation and mutation.

There are several attractive features of this hypothesis. The conformational profile of Gma5 and Gmi3 are relatively similar (Figure 8Go) and resemble those of a DNA base more than Gma3 (data not shown) or Gmi5 (Figure 8Go). The hypothesis provides at least an incipient rationale for what the difference might be between induction of a G->T versus a G->A mutation by [+ta]-B[a]P–N2-dG; perhaps these base displaced conformations are recognized as non-coding and when there is no bulk in the minor groove, then DNA polymerase is triggered to insert dATP, while if there is bulk in the minor groove, then dTTP is inserted. (The reasons for such preferences are not obvious.)

Some of our experimental results forced us to propose that the conformation responsible for a G->T mutation must be relatively different than for a G->A mutation, since interconversion between them must be slow (see Introduction and above; 10,13,14,16,17). The pathways depicted in Figure 1Go provide at least a potential rationale for this, in that Gma5 and Gmi3 are separated from each other by at least four steps, any one of which might be relatively slow. Certainly, it is not unreasonable to imagine that the complex process that must occur in order for Gma5 to interconvert to Gmi3, which involves the dG moiety switching grooves and the B[a]P moiety flipping over, might be relatively slow, especially given that a DNA polymerase appears to surround DNA in its active site and hold it relatively rigidly (31). If this is true, then the `decision point' (Figure 4Go) for whether the G->T versus G->A mutational pathway is followed might be made at the point when DNA polymerase encounters the lesion and, thus, mutagenesis is dictated by the conformation of the adduct in dsDNA, even though the actual mutagenic event does not occur until later in the pathway. Some of our experimental results (10,16) suggested that frameshift and base substitution mutational pathways might involve conformations that do interconvert relatively rapidly and, therefore, might be closer than (for example) the G->T versus G->A mutational pathway; it is of interest that the frameshift conformations are separated from base substitution conformations by a single step (Figure 6Go).

Finally, it is important to note that the putative mutagenic conformations, Gma5 and Gmi3, are calculated to be relatively minor conformations compared with the putative non-mutagenic conformations BPmi5 and BPmi3 in most cases (21), but this is not inconsistent with mutagenesis being a relatively infrequent event.


    Notes
 
1 To whom correspondence should be addressed Email: loechler{at}bio.bu.edu Back


    Acknowledgments
 
We thank the Scientific Computing and Visualization group at Boston University for allocations of computational resources. This work was supported by NIH grant CA50432.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received June 10, 1998; revised August 24, 1998; accepted September 2, 1998.