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The Transversion Pathway (Spontaneous Mutation/Acid Mutagenesis/Glycosidic Bond Migration) PAUL M

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The Transversion Pathway (Spontaneous Mutation/Acid Mutagenesis/Glycosidic Bond Migration) PAUL M Proc. Natl. Acad. Sci. USA Vol. 73, No. 11, pp. 4159-4163, November 1976 Genetics Heat mutagenesis in bacteriophage T4: The transversion pathway (spontaneous mutation/acid mutagenesis/glycosidic bond migration) PAUL M. BINGHAM, RICHARD H. BALTZ*, LYNN S. RIPLEY, AND JOHN W. DRAKEt Department of Microbiology, University of Illinois, Urbana, Ill. 61801 Communicated by Matthew S. Meselson, September 3, 1976 ABSTRACT Heat induces transversions (as well as transi- produce the homologous ochre (UAA) mutant and therefore tions) at GC base pairs in bacteriophage T4. The target base for go undetected, the heat-induced reversion of amber mutants transversions is guanine, which is converted to a product which could occur only by transversions at the G-C base pair (pro- is sometimes replicated and transcribed as a pyrimidine. A model for this process is proposed in which the deoxyguanosine ducing the tyrosine codons UAU or UAC). Table 2 shows that glycosidic bond migrates from N9 to N2: the resulting deoxy- amber mutants readily revert when heated. neoguanosine may pair with normal guanine to produce GC Amber mutants might, however, revert by extracistronic _ CG transversions. suppressors arising by G-C A-T transitions. Hydroxylamine specifically induces this transition, and a considerable number We have previously described the induction by heat of G-C - of amber mutants in the WI region have already been shown A-T transitions in bacteriophage T4 by deamination of cytosine to be refractory to hydroxylamine-induced reversion (3, 4). We to produce uracil (1). Earlier studies of heat mutagenesis in have extended this result by showing that the amber mutants bacteriophage T4 suggested that transversions were also in- described in Table 2 are also insusceptible to hydroxylamine- duced at G-C base pairs (2), and this conjecture is now con- induced reversion even when the treated stocks are grown firmed. We have also confirmed previous results indicating that under permissive conditions before selective plating for the rII+ neither base pair substitutions at A-T sites nor frameshift revertants. (Hydroxylamine-treated HB129AM produces a few mutations are induced by heat mutagenesis. revertants that are not wild type, but these are readily distin- guishable from the revertants of normal appearance induced MATERIALS AND METHODS by heat.) Most materials and methods have been described previously Guanylate Residues Are the Mutagenic Targets for the (ref. 1 and references therein). Drake top and bottom agars Transversion Pathway. Among the revertants of rII mutants were usually used, but enriched H agar was used in experiments arising as rII/r + heteroduplex heterozygotes, only those con- involving amE509; supersoft enriched H top agar contained taining the r+ allele on the transcribed strand are detected by only 0.42% agar. Escherichia coli was used throughout: strain direct plating on a nonpermissive host; those containing the r+ BB to grow and titer T4 wild-type and rnI strains and to score allele on the complementary strand must be grown for one cycle revertants of amE509, strain KB to score rnI + revertants, and ("passaged") on a permissive host before revertants can be strain CR63 (an amber suppressor) to grow and titer amE509. detected by plating on a nonpermissive host. At the G-C site of All T4rII mutants are in a T4B background; amE509 is in a an amber (UAG) mutant the transcribed strand contains the T4D background. Phage stocks were purified by differential cytosine and the complementary strand the guanine residue. centrifugation and resuspended in 1 mM sodium phosphate, Table 2 shows that heat-induced reversion of rnI amber mutants pH 7.0, plus 0.5 mM MgCI2, except that the stocks used in the is detected mainly or exclusively only after passaging, thus experiments described in Tables 1 and 2 (excluding those entries implicating the guanylate residue as the primary mutagenic in Table 1 quoted from previous work) were resuspended di- target for the transversion pathway. [Cytosine is the mutagenic rectly in the heating buffers. During heating, pH was deter- target for the transition pathway (1).] mined under experimental conditions, and heating was ter- The alternative hypothesis, that cytosine is the mutagenic minated by dilution into chilled 100 mM sodium phosphate, target in the transversion mechanism, is contradicted by the pH 7.0, plus 0.5 mM MgCl2. behavior of the rII missense mutant rUV74 (Table 2). rUV74 is insusceptible to reversion by mutagens producing transitions RESULTS (base analogues, hydroxylamine), and its heat-induced rever- Neither Base Pair Substitutions at A-T Sites nor Frameshift tants are expressed upon direct plating on the nonpermissive Mutations Are Induced by Heat. Data accumulated to date host. For cytosine to be the target in a reverting amber (UAG) on the reversion responses of T4rII mutants reverting only at codon, its heat-induced derivative would have to be replicated A-T site, or only by frameshift mutagenesis, are summarized as a purine, but transcribed either not at all or else as a pyrim- in Table 1. None of 15 A-T and 10 frameshift mutants was in- idine. In that case, no nIl mutant could exhibit heat-induced duced to revert by heat under a variety of conditions, even transversion reversion upon direct plating, whereas rUV74 when heated stocks were grown under permissive conditions does. before selective plating to detect rnI + revertants. Thus, the guanylate residue is the target for the heat-induced Heat Induces Transversion Mutations at G-C Sites. Since transversion and the product of the mutagenic reaction can be A-T base pairs are refractory to heat mutagenesis, and since transcribed and replicated as a pyrimidine. an mutant The Transversion Pathway May Be GC -- CG. transitions at the G-C base pair of amber (UAG) Transversion mutations arising at G-C sites could produce either * Present address: Biochemical Development Division, Eli Lilly and C-G or T-A base pairs. At present we still lack tester strains ca- Co., Indianapolis, Ind. 46206. pable of distinguishing between these two pathways. However; t To whom communications should be addressed. a previous collection (2) of heat-induced rnl mutants consisted, 4159 Downloaded by guest on September 30, 2021 4160 Genetics: Bingham et al. Proc. Nati. Acad. Sci. USA 73 (1976) Table 1. Failure of A- T and frameshift mutants to revert Table 2. Induction of G- C-site transversions by heat when heated Revertants per 108 survivorst Revertants per 108 survivorst H(-)- H(-)- H(+) H(+)- H(-)- H(-)- H(+)- H(+)- Mutant* Seriest P(-) P(+) P(-) P(+) Mutant* Seriest P(-) P(+) P(-) P(+) rHB129AM VII 6 9 15 63 A T mutants rP7AM VIII 0.7 0.5 < 0.9 6.5 42 rP70C VI 2 2 2 3 rUV200AM IV 3 7 4 rSM120C VI 9 7 10 7 V 6 9 9 56 rSM41 OC VI 9 11 10 10 VI 8 6 3 30 rUV16 I 26 18 rUV74 IV 35 50 700 460 rUV69 I 0.1 0.1 * The AM suffix indicates an amber (UAG) mutant. The three am- rUVI1 7 II 2000 2000 ber mutants (but not rUV74) are revertible by base analogues, III 2000 1700 but none of the mutants is revertible by hydroxylamine, even IV 2500 4400 1700 4000 after passaging. V 2000 3800 1700 4800 t IV, V, and VI are described in a footnote to Table 1. VII = 26 hr rUVI99 II 23 20 at 600 in 10 SmM sodium phosphate, pH 7.0, plus 0.5 mM MgCl2. III 23 25 VIII = 28 hr at 570 in 1 mM sodium phosphate, pH 7.0, plus 0.5 IV - 320 400 440 440 mM MgCl2. 1. V 150 390 200 11:u t See footnote to Table rUV2000C IV 1 1 3 3 V 3 4 2 2 after the frameshift mutants contributed VI 3 1 3 3 proflavin-revertible rUV215 IV 35 52 50 20 by the spontaneous background was discarded, of two classes. V 21 24 33 17 The members of one class were revertible by base analogues and rUV236 V 50 130 74 92 might, therefore, have arisen by the G-C , A-T transitions rUV304 I 1 1 already known to be induced by heat (1). However, many of V 13 33 21 these mutants were also revertible by hydroxylamine, which rUV354 I 8 8 specifically induces G-C -- A-T transitions. It was therefore rUV3570C VI 5 7 5 proposed that these mutants were induced by G-C - C-G rUV373 II 2300 2800 transversions, but are revertible to T-A by hydroxylamine, III 2300 2800 producing an acceptable but usually nonwild-type amino acid rUV379 I 0.5 0.5 in the rIl polypeptide. Frameshift mutants The members of the second class were revertible by neither nor These mutants might have arisen rUV2 I 52 73 base analogues proflavin. IV 110 67 150 either by transversion mutagenesis, generating a codon inca- rUV20 I 4 4 pable of reversion along a transition pathway, or by frameshift rUV27 II 1200 1400 mutagenesis, generating mutants nevertheless insusceptible to III 1200 1200 proflavin-induced reversion. [Note that of 12 proflavin-induced IV 6500 6500 5000 rII mutants, four were nonrevertible by proflavin, 5-amino- V 6800 7500 4300 acridine, or both (5).] We therefore further examined five rUV28 IV 74 90 92 mutants of this class, chosen for their varied reversion rates. The rUV34 I 1900 2500 IV 1500 2400 1000 revert by A-T - G.C transitions and sometimes by A.T-site rUV81 I 1.5 <1 transversions, but not (according to the conclusions of this report) I 1 <1 by G.C-site transversions. rP70C and rUV117, however, revert rUV1 03 only by A-T-site transversions (25).
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