A molecular characterization of spontaneous frameshift mutagenesis within the trpA gene of Escherichia coli

Abstract

Spontaneous frameshift mutations are an important source of genetic variation in all species and cause a large number of genetic disorders in humans. To enhance our understanding of the molecular mechanisms of frameshift mutagenesis, 583 spontaneous Trp+ revertants of two trpA frameshift alleles in Escherichia coli were isolated and DNA sequenced. In order to measure the contribution of methyl-directed mismatch repair to frameshift production, mutational spectra were constructed for both mismatch repair-proficient and repair-defective strains. The molecular origins of practically all of the frameshifts analyzed could be explained by one of six simple models based upon misalignment of the template or nascent DNA strands with or without misincorporation of primer nucleotides during DNA replication. Most frameshifts occurred within mononucleotide runs as has been shown often in previous studies but the location of the 76 frameshift sites was usually outside of runs. Mismatch repair generally was most effective in preventing the occurrence of frameshifts within runs but there was much variation from site to site. Most frameshift sites outside of runs appear to be refractory to mismatch repair although the small number of occurrences at most of these sites make firm conclusions impossible. There was a dense pattern of reversion sites within the trpA DNA region where reversion events could occur, suggesting that, in general, most DNA sequences are capable of undergoing spontaneous mutational events during replication that can lead to small deletions and insertions. Many of these errors are likely to occur at low frequencies and be tolerated as events too costly to prevent or repair. These studies also revealed an unpredicted flexibility in the primary amino acid sequence of the trpA product, the alpha subunit of tryptophan synthase.

Fig. 1

The nucleotide and amino acid changes associated with the trpA9813, trpA540, and trpA21 frameshift mutations and reversion frequencies of these alleles are shown below. The pertinent portions of the trpA gene and its product the alpha subunit of tryptophan synthase are given. The numbers for the gene and amino acid sequence correspond to those used by Nichols and Yanofsky [27]. The nucleotide deleted in each frameshift is boxed. For the trpA540 and trpA21 alleles, the single C deleted can be any in a run of four.

Fig. 2

Proposed mechanisms (listed in Table 4) for the occurrence of representative frameshift revertants: (a) Simple slippage of the nascent strand in mononucleotide runs; (b) Misincorporation of a primer nucleotide followed by misalignment of the nascent strand; (c) Misincorporation of the primer nucleotide followed by misalignment of the template strand; (d) Misalignment of the nascent strand with no misincorporation of a primer nucleotide; (e) Misalignment of the template strand with no misincorporation of a primer nucleotide; and (f) Misincorporation of a primer nucleotide followed by correct incorporation of the next nucleotide followed by misalignment of the nascent strand. For most frameshift events, except for 2f, either DNA strand could be the primer strand. In these cases the choice of primer and template strands for the models was based upon the following: (1) A transversion mispairing is assumed to cause the DNA polymerase complex to stall longer at the mismatch than a transition mismatch, providing a greater opportunity for misalignment to occur [32] and (2) AT-richer sequences in newly synthesized DNA provide greater instability leading to misalignment. (See text for further detail on these models.)

Fig. 3

Possible mechanisms (called ‘complex’ in Table 4) to account for frameshift revertants not explained by the models of Fig. 2. (a) Misincorporation of the primer nucleotide followed by a second misincorporation of the next primer nucleotide and finally misalignment of the primer strand (shown here for trpA9813 revertant R18). (b) Creation of a hairpin loop followed by either by precise excision of the single-stranded region (trpA9813 R8) or imprecise excision (trpA9813 R13). As shown here the formation and excision of the hairpin loop is occurring during replication but could occur anytime this region is single-stranded.

Fig. 4

The composite amino acid replacements fortrpA9813 and trpA540 revertants are shown below. All the amino acids found at each position that differ from the mutant peptide sequence are included as well as deletions and insertions. A “+” indicates the insertion of a single amino acid at a position, “++” the insertion of two amino acids, and “−” a deletion. The 175, 177, 179, 211, and 213 sites were shown to be critical for enzymatic activity in previous studies (see text). Revertants oftrpA9813 had wild-type sequences before position 172 and after 179; trpA540 revertants had wild-type sequences before position 201 and after 224.