Demonstration of the production of frameshift and base-substitution mutations by quasipalindromic DNA sequences.

Abstract

The in vivo production of frameshift and base-substitution mutations predicted as a consequence of the metabolic processing of misaligned quasipalindromic DNA sequences has been confirmed. Spontaneous frameshift mutations of the T4 Wi gene that had been genetically mapped to quasipalindromic DNA sequences were sequenced. Some of the mutant sequences are exactly those predicted by a mutational model based on misaligned quasipalindromes. Furthermore, these sequences are distinct from those predicted by the classical frameshift model based on misaligned repeated sequences. The ilI frameshift mutant sequences reported here result from the deletion of a specific base or bases that would remain looped out should the quasipalindromes assume a hairpin secondary structure. One hairpin predicted not only the deletion of two bases (a frameshift) but the concomitant production of nearby but noncontiguous base substitutions. The substitution of as many as three bases as well as the frameshift were predicted to arise as a consequence of a single mutational event in the palindrome. Two independent examples of the predicted deletion frameshift were found among the small sample of sequenced spontaneous frameshifts examined and both were associated with the predicted transversion and transition base substitutions.Frameshift mutations often result from the addition or deletion of a repeating unit of a repeated DNA sequence. Metabolic processing of misaligned pairing between one of the repeating units and the complement of another is believed to be responsible (1, 2). However, frameshifts can occur in DNA sequences that are not repeated. A consideration of molecular mechanisms responsible for these mutations led to the proposal that some frameshifts occur because of processing of misaligned pairing mediated by quasipalindromes (3-5).Palindromic sequences permit the formation of alternative DNA secondary structures such as DNA hairpins. The action of ordinary DNA metabolism on such structures is predicted to lead to mutation (3, 5). The demonstration of mutations predicted to occur as a consequence of such processing and not by other mutational models would be strong confirmation of the quasipalindrome mutational model and would suggest the occurrence of palindromically mediated misalignments in vivo.Quasipalindromes permit the formation of misaligned structures through the self-complementarity of their palindromic portions and provide templates for base-substitution and frameshift mutations in their nonpalindromic portions (3). Mutations are predicted to result from templated processes that replace bases residing in one half of the hairpin with bases complementary to the other half. The predicted mutations can be readily visualized by examining quasipalindromic DNA sequences in hairpin structures (Fig. 1). Each unpaired base in the stem of the hairpin lacks a complemen-

Insights Into Mutagenesis Using Escherichia coli Chromosomal lacZ Strains That Enable Detection of a Wide Spectrum of Mutational Events

“…An important class of mutational hotspots includes short imperfect inverted repeat sequences, also known as quasipalindromes (QPs) (Figure 2A). These can be seen in mutational spectra in bacteriophage, bacteria, yeast, and in mutations responsible for several human genetic diseases (Stewart and Sherman 1974;Ripley 1982;De Boer and Ripley 1984;Mo et al 1991;Demarini et al 1993;Cebula 1995;Greenblatt et al 1996;Bissler 1998;Yoshiyama and Maki 2003;Schultz et al 2006). Genetic analysis of the mechanism of mutagenesis suggests that these hotspot mutations, which always improve the perfection of the inverted repeat sequence, occur by template-switch reactions during normal replication (Viswanathan et al 2000;Dutra and Lovett 2006), as proposed initially by Ripley (1982).…”

“…The formation of an intrastrand hairpin structure on the nascent strand produces an alternative template for synthesis leading to mutations ( Figure 2B). Alternatively, strand displacement and annealing across the replication fork at the site of the inverted repeats provides a template for synthesis of the hotspot mutation ( Figure 2C) (Ripley 1982;De Boer and Ripley 1984;Rosche et al 1995Rosche et al , 1997Rosche et al , 1998. These template-switch reactions have been postulated to occur after stalled replication since mutations in E. coli SOS-induced translesion polymerases elevate hotspot mutagenesis in the thyA gene (Dutra and Lovett 2006).…”

Mismatch repair in recombination of bacteriophage T4

“…Studies of T4 rIIB mutants (94,95) provided evidence for in vivo production of mutations via metabolic processing of quasipalindromic DNA sequences. Because mismatched regions in the stem-loop or hairpin structures formed by quasipalindromic sequences are similar to those in recombination heteroduplexes, it is reasonable to think that they are processed by the same mechanism.…”

Multiple substitutions create biased estimates of divergence times and small increases in the variance to mean ratio

“…Mutations that are easily explained by misalignment mechanisms have been observed in the phage T4 (deBoer and Ripley, 1984), in B. coli, in Salmonella typhimurium and in yeast . Complementary to these studies, exciting in vitro experiments are showing a necessary role for direct repeats in the production of base substitutions and deletions (Kunkel and Alexander, 1985).…”