Lynn S. Ripley

Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase☆

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.

Spectrum of spontaneous frameshift mutations: Sequences of bacteriophage T4 rII gene frameshifts☆

The DNA sequences of 185 independent spontaneous frameshift mutations in the rIIB gene of bacteriophage T4 are described. Approximately half of the frameshifts, including those at hot spot sites, are fully consistent with classical proposals that frameshift mutations are produced by a mechanism involving the misaligned pairing of repeated DNA sequences. However, the remaining frameshifts are inconsistent with this model. Correlations between the positions of two base-pair frameshifts and the bases of DNA hairpins suggest that local DNA topology might influence frameshift mutation. Warm spots for larger deletions share the property of having endpoints adjacent to DNA sequences whose complementarity to sequences a few base-pairs away suggest that non-classical DNA misalignments may participate in deletion mutation. A model for duplication mutation as a consequence of strand displacement synthesis is discussed. In all, 15 frameshifts were complex combinations of frameshifts and base substitutions. Three of these were identical, and have extended homology to a sequence 256 base-pairs away that is likely to participate in the mutational event; the remainder are unique combinations of frameshifts and transversions. The frequency and diversity of complex mutants suggest a challenge to the assumption that the molecular evolution of DNA must depend primarily upon the accumulation of single nucleotide changes.

Mutator versus antimutator activity of a T4 DNA polymerase mutant distinguishes two different frameshifting mechanisms

SummaryClassical “antimutator” DNA polymerases of bacteriophage T4 were examined for their effects upon frameshift mutation rates at a number of positions within rII cistrons. Their antimutagenic activities reduced frameshift frequencies at a number of sites, but at other sites the opposite occurred: the mutant polymerases exhibited clear mutator activities. This dichotomy reveals the operation of two distinct mechanisms of frameshift mutagenesis that are correlated with the DNA sequences at the frameshift sites. Frameshift mutants subject to the antimutator effects of the mutant polymerase lie in A:T-run DNA sequences, where mutations presumably arise by means of the interstrand DNA misalignments postulated by classical theory. The frameshift mutants produced by the mutator activity of these same polymerases lie in quasipalindromic DNA sequences, where mutations are postulated to arise by aberrant metabolism of DNA secondary structures such as hairpins.

Estimation of in-vivo miscoding rates.

The replication of premutagenic DNA lesions generates mutant progeny in patterns that distinguish lesions that rarely produce a mutation per DNA replication from those that frequently do so. The quantitative aspects of this distinction were tested in studies of heat-mutagenized bacteriophage T4. Previous T4 studies had demonstrated that transition mutations produced at G.C base-pairs depended upon heat-induced DNA lesions distinct from those responsible for transversions at G.C pairs. In this study the transversion mutations are shown to arise in patterns predicted for mutations produced from lesions that miscode rarely (fewer than 10% per replication). In contrast, the transition mutations arise in patterns predicted for mutations produced from lesions that miscode at about 20 to 60% per replication. The fact that the two classes of DNA lesions are distinguishable as predicted by the quantitative model suggests that such studies may in general be useful in quantifying the behavior of mutation-generating DNA lesions. The method employed also estimates the frequency of premutagenic lesions in DNA.

Influence of diverse gene 43 DNA polymerases on the incorporation and replication in vivo of 2-aminopurine at A · T base-pairs in bacteriophage T4

The relative rates at which A · T → AP · T† base-pairs are formed in DNA during 2-aminopurine mutagenesis, and the relative rates at which AP · T → AP · C base-pairs are formed during the in vivo replication of templates containing 2-aminopurine, were measured for a mutator, a wild type, and two antimutator bacteriophage T4 DNA polymerases. These measurements did not require quantitation of the 2-aminopurine in the T4 DNA. The mutator polymerase had no discernible effect upon the rate of A · T → AP · T, but increased the rate of AP · T → AP · C twofold over that of the wild-type enzyme. The two antimutator polymerases decreased the rate of formation of both AP · T and AP · C base-pairs compared to the wild-type enzyme. The relative antimutator effect is asymmetrically divided between the two mutation steps producing a very large decrease in the AP · T → AP · C step but a much smaller decrease in the A · T → AP · T step. This and additional observations suggest that the fidelity-enhancing components of T4 DNA polymerases distinguish between 2-aminopurine in template DNA and 2-aminopurine at the primer terminus.

Bacteriophage T4 particles are refractory to bisulfite mutagenesis.

Bisulfite-induced deamination of cytosine produces uracil, a thymine analog reported to be mutagenic both in vitro and in vivo. Although deamination of cytosine in DNA should produce G:C----A:T transitions, treating bacteriophage T4 particles with 0.9 M bisulfite at pH 5 at 37 degrees C produced no more mutations than did the equivalent buffer without bisulfite. Lack of bisulfite mutagenicity is fully consistent with the reported resistance of 5-substituted cytosines to bisulfite-induced deamination, since T4 DNA contains glucosylated 5-hydroxymethylcytosine. However, bisulfite also failed to induce mutations in T4 particles whose DNA contained unmodified cytosine. The lack of mutagenesis persisted in E. coli hosts deficient in uracil glycosylase, an enzyme expected to participate in the repair of the putative bisulfite-generated uracil. Cytosine in T4 DNA may be largely protected from bisulfite attack within phage particles.

Polymerase Infidelity and Frameshift Mutation

Mutant T4 DNA polymerases which alter mutation rates in vivo have been used to approach questions of replication fidelity. Most studies have characterized “mutator” or “antimutator” polymerases by their influence upon base-pair substitution mutation, particularly transitions. We are extending the characterization of mutant polymerases to include the role of T4 DNA polymerase in frame fidelity.

The Specificity of Infidelity of DNA Polymerase

A complex network of metabolic processes is responsible for the accurate production of progeny DNA. The infidelity of these processes when measured as mutations per base pair in DNA varies dramatically among organisms (1) and seems likely to reflect unique combinations of fidelity determinants. Unfortunately, it is nearly impossible to measure fidelity directly, but instead it is the summation of accurate and inaccurate processes producing a mutation frequency which can be experimentally determined.

Mutagenic specificity of nitrosomethylurea in bacteriophage T4

In contrast to alkylating agents such as ethyl methanesulphonate which can induce mutation after treatment of free phage particles, nitrosamides induce mutations only when phage are treated intracellularly during infection of the host. The basis of this intracellular dependence is not currently understood. In this study the mutational specificity of nitrosomethylurea (NMU) in bacteriophage T4 was investigated by measuring the reversion of well-characterized mutants in the rII genes. While no mutation was produced after in vitro treatments of free phage, in vivo treatments strongly induced G:C leads to A:T transitions and substantially induced A:T leads to G:C transitions. Transversions and frameshift mutations were rarely induced. Although methyl methanesulfonate mutagenesis of T4 depends upon a phage-encoded error-prone repair system, NMU-induced mutagenesis is independent of this repair system. Similarities in mutagen specificity of nitrosamides and differences in DNA metabolism in T4 when compared to its host, Escherichia coli, suggest that T4 is well-suited for the study of mechanisms of nitrosamide mutagenesis.