Reversion of Frameshift Mutations by Mutator Genes in Escherichia coli

The Escherichia coli mutator genes mutU4, mutS3, and mut-25 (a possible allele of mutL), previously known to induce transitional base changes, increased significantly the frequencies of reversion of lacZ frameshift mutations. mutT1, previously shown to induce only the transversion of adenine-thymine to cytosine-guanine, had no effect on the reversion of lacZ frameshift mutations. With mutator genes other than mutT1, small increases were found in the frequencies of reversion of trpA frameshift mutations.     

Opposing Roles of the Holliday Junction Processing Systems of Escherichia Coli in Recombination-Dependent Adaptive Mutation

Aspects of the molecular mechanism of ``adaptive' mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid. Homologous recombination is required and the mutations resemble polymerase errors. Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon. Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other. Moreover, temporary absence of both resolution systems promotes mutation. These results imply that recombination intermediates themselves promote the mutations.     

Spontaneous Mutation in the Escherichia Coli Laci Gene

To gain more detailed insight into the nature and mechanisms of spontaneous mutations, we undertook a DNA sequence analysis of a large collection of spontaneous mutations in the N-terminal region of the Escherichia coli lacI gene. This region of circa 210 base pairs is the target for dominant lacI mutations (i-d) and is suitable for studies of mutational specificity since it contains a relatively high density of detectable mutable sites. Among 414 independent i-d mutants, 70.8% were base substitutions, 17.2% deletions, 7.7% additions and 4.3% single-base frameshifts. The base substitutions were both transitions (60%) and transversions (40%), the largest single group being G.C----A.T (47% of base substitutions). All four transversions were observed. Among the 71 deletions, a hotspot (37 mutants) was present: an 87-bp deletion presumably directed by an 8-bp repeated sequence at its endpoints. The remaining 34 deletions were distributed among 29 different mutations, either flanked (13/34) or not flanked (21/34) by repeated sequences. The 32 additions comprised 29 different events, with only two containing a direct repeat at the endpoints. The single-base frameshifts were the loss of a single base from either repeated (67%) or nonrepeated (33%) bases. A comparison with the spectrum obtained previously in strains defective in DNA mismatch correction (mutH, mutL, mutS strains) yielded information about the apparent efficiency of mismatch repair. The overall effect was 260-fold but varied substantially among different classes of mutations. An interesting asymmetry was uncovered for the two types of transitions, A.T----G.C and G.C----A.T being reduced by mismatch repair 1340- and 190-fold, respectively. Explanations for this asymmetry and its possible implications for the origins of spontaneous mutations are discussed.     

A Temperature Sensitive Suppressor Mutation of Escherichia Coli

Abstract

In an Escherichia coli K 12 strain, with a Pho ^- nonsense mutation and the suppressor gene Su-4, we have induced a mutant with a temperature dependent alkaline phosphatase negative phenotype. This phenotype can be ascribed to a mutation mapping in the Su-4 gene that makes the suppression temperature sensitive.     

A Set of Lacz Mutations in Escherichia Coli That Allow Rapid Detection of Specific Frameshift Mutations

We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites.     

Two Enzymes, Both of Which Process Recombination Intermediates, Have opposite Effects on Adaptive Mutation in Escherichia Coli

Reversion of a lac(-) frameshift allele carried on an F' episome in Escherichia coli occurs at a high rate when the cells are placed under lactose selection. Unlike Lac(+) mutations that arise during nonselective growth, the production of these adaptive mutations requires the RecA-RecBCD pathway for recombination. In this report, we show that enzymes that process recombination intermediates are involved in the mutagenic process. RuvAB and RecG, E. coli's two enzymes for translocating Holliday junctions, have opposite effects: RuvAB is required for RecA-dependent adaptive mutations, whereas RecG inhibits them.     

Levels of the Vsr Endonuclease Do Not Regulate Stationary-Phase Reversion of a Lac− Frameshift Allele in Escherichia coli

Vsr endonuclease, which initiates very short patch repair, has been hypothesized to regulate mutation in stationary-phase cells. Overexpression of Vsr does dramatically increase the stationary-phase reversion of a Lac⁻ frameshift allele, but the absence of Vsr has no effect. Thus, at least in this case, Vsr has no regulatory role in stationary-phase mutation, and the effects of Vsr overproduction are likely to be artifactual.     

Evidence for Frameshift Mutations in the Hish Gene of Escherichia Coli Causing Synthesis of a Partially Active Glutamine Amidotransferase

Among eight strains carrying acridine-induced mutations in hisH, five which mapped at four different sites in the promoter-distal region of the gene showed His+ phenotypes on media containing a purine. By complementation analysis, hisH enzyme was shown to be required for growth on purines. Purine-sensitive His+ revertants of strains able to grow on purines carried second-site mutations which in one case could be shown to map in hisG. Strains able to grow on purines were able to grow on 2-thiazolyl-DL-alanine, too. We conclude that frameshift mutations in the promoter-distal part of the hisH gene of E. coli do not completely abolish the activity of the gene product.     

Reversion of the Frameshift hisD3018 of Salmonella typhimurium

Previous studies on histidinol dehydrogenase from His(+) revertants have shown that the frameshift mutation hisD3018 is a +1 type, resulting from inclusion of an extra cytidylate residue in messenger ribonucleic acid. Histidinol dehydrogenase from newly isolated spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced intragenic revertants has been examined for amino acid replacements. The results provide additional evidence that NG can delete guanine plus cytosine base pairs from deoxyribonucleic acid. One spontaneous revertant was found to result from a +2 addition of approximately 16 nucleotide residues before the +1 parent frameshift, and another by a -4 deletion about six residues before the same. Circumstantial evidence suggests the in vivo codon assignment GAG for glutamic acid. A region of histidinol dehydrogenase highly permissive of amino acid changes encoded in the minus (-) phase is now apparent.     

Spontaneous Mutation in Escherichia Coli Containing the Dnae911 DNA Polymerase Antimutator Allele

We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants have a mutation in the dnaE gene, encoding the α subunit of DNA polymerase III. They were isolated in a mismatch-repair-defective mutL background, in which mutations can be considered to represent uncorrected DNA replication errors. In the present study we analyze the effect of one of these alleles, dnaE911, on spontaneous mutagenesis in a mismatch-repair-proficient background. In this background, spontaneous mutations may be the sum of uncorrected replication errors and mutations resulting from other pathways. Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation. The data show that dnaE911 decreases the level of Rif(r), lacI and galK mutations in this background by 1.5-2-fold. DNA sequencing of 748 forward mutants in the lacI gene reveals that this effect has a clear specificity. Transversions are decreased by ~3-fold, whereas transitions, frameshifts, deletions and duplications remain essentially unchanged. Among the transversions, A·T -> T·A are affected most strongly (~6-fold). In addition to this effect on transversions within the lacI gene, one previously recognized A·T -> G·C base-pair substitution hotspot in the lac operator is also reduced (~5-fold). The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.     

Nature of the hisD3018 Frameshift Mutation in Salmonella typhimurium

Histidinol dehydrogenase from three differing revertants of ICR-191A-induced frameshift hisD3018 has been purified and examined for amino acid replacements. The enzyme from one spontaneously arising revertant, R7, contains an extra proline residue, whereas that from another, R5, contains an extensive frameshifted sequence, four amino acid residues of which have been identified to date. The amino acid replacement data are in agreement with the in vitro code word assignments and allow the characterization of the hisD3018 frameshift as an addition of one nucleotide pair, most likely guanine plus cytosine. Enzymatic data for those ICR-191A-induced revertants of hisD3018 arising within the hisD gene indicate that the enzyme is wild type and, therefore, that ICR-191A can cause deletions as well as additions of single base pairs. The wild-type amino acid sequence is restored in enzyme from an N-methyl-N′-nitro-N-nitrosoguanidine (NG)-induced revertant, R29, suggesting that NG is a base-deleting as well as a base-substituting mutagen. The unusual response of hisD3018 to external suppressors is considered in terms of reinitiation of protein synthesis out of phase, coupled with suppression of a nonpermissive missense codon so generated, and of an alternative hypothesis invoking a true frameshift suppressor transfer ribonucleic acid with an extended or deleted anticodon.     

Reversion of a streptomycin-dependent strain of Escherichia coli

Summary A streptomycin dependent, spectinomycin resistant mutant ofEscherichia coli was used to select spontaneous phenotypic revertants to non-dependence on streptomycin. The ribosomes from one such revertant, which is inhibited by both streptomycin and spectinomycin, were analyzedin vitro. The altered protein responsible for the suppression of the streptomycin dependent phenotype was identified; this protein is 30S-10. The genetic locus for this mutation is a newly identified locus and it has been positioned close to thestr locus. The identification of the altered component responsible for the suppression of the spectinomycin resistant phenotype may be the same as that for the streptomycin dependent phenotype, but this is unproven.     

Duplication Mutation as an Sos Response in Escherichia Coli: Enhanced Duplication Formation by a Constitutively Activated Reca

The SOS response in Escherichia coli involves the induction of a multioperon regulatory system, which copes with the presence of DNA lesions that interfere with DNA replication. Induction depends on activation of the RecA protein to cleave the LexA repressor of SOS operons. In addition to inducible DNA repair, the SOS system produces a large increase in the frequency of point mutations. To examine the possibility that other types of mutations are induced as part of the SOS response, we have studied the production of tandem duplications. To avoid the complications of indirect effects of the DNA lesions, we have activated the SOS response by a constitutive mutation in the recA gene, recA730. The introduction of the recA730 mutation results in an increase in duplications in the range of tenfold or greater, as judged by two different criteria. Based on its genetic requirements, the pathway for induced duplication formation is distinct from the point mutation pathway and also differs from the major normal recombination pathway. The induction of pathways for both duplications and point mutations shows that the SOS system produces a broad mutagenic response. We have suggested previously that many types of mutations might be induced by severe environmental stress, thereby enhancing genetic variation in an endangered population.