Four-base codons ACCA, ACCU and ACCC are recognized by frameshift suppressor sufJ

The frameshift suppressor sufJ acts to correct a set of +1 frameshift mutations having very different sequences at their mutant sites. This suppressor acts by reading a 4 base codon located near, but not at, the site of each suppressible mutation. Suppression thus necessitates out-of-phase translation of the short stretch of mRNA between the site of action of the suppressor tRNA and the site of the frameshift mutation. We have identified the site read by sufJ by mutationally creating a series of such sites in the neighborhood of a previously nonsuppressible frameshift mutation. Each of the newly generated sites was formed by base substitution. Four independently generated sites were analyzed by DNA sequencing. At each site the quadruplet codon ACCX was generated (where X is A, U or C). Thus sufJ is able to read a 4 base codon in which any of three bases is acceptable in the fourth position. This is the first frameshift suppressor that does not read a run of three repeated bases in the first three positions of its codon.     

Polymerase-specific differences in the DNA intermediates of frameshift mutagenesis. In vitro synthesis errors of Escherichia coli DNA polymerase I and its large fragment derivative

The sequences of more than 600 frameshift mutations produced as a consequence of in vitro DNA replication on an oligonucleotide-primed, single-stranded DNA template by the Escherichia coli polymerase I enzyme (PolI) or its large fragment derivative (PolLF) were compared. Four categories of mutants were found: (1) single-base deletions, (2) base substitutions, (3) multiple-base deletions and (4) complex frameshift mutations that change both the base sequence and the number of bases in a concerted mutational process. The template sequence 5'-Py-T-G-3', previously identified as a PolLF hotspot for single-base deletions opposite G, is also a hotspot for PolI. A PolI-specific warm spot for single-base deletions was identified. Among base substitutions, transitions were more frequent than transversions. Transversions were mediated by (template)G.G, (template)G.A, and (template)C.T mispairs. Multiple-base deletions were found only after PolI replication. Although each of these deletions can be explained by a misalignment mediated by directly repeated DNA sequences, deletion frequencies were often different for repeats of the same length. Both PolI and PolLF produced many complex frameshift mutants. The new sequences at the mutant sites are exactly complementary to nearby DNA sequences in the newly synthesized DNA strand. In each case, palindromic complementarity could mediate the misalignment needed to initiate the mutational process. The misaligned DNA synthesis accounts for the nucleotide changes at the mutant site and for homology that could direct realignment of the DNA onto the template. Most of the complex mutant sequences could be initiated by either intramolecular misalignments involving fold-back structures in newly synthesized DNA or by strand-switching during strand-displacement synthesis. The striking differences between the specificities of complex frameshift mutations and multiple-base deletions by PolI and PolLF identify the existence of polymerase-specific determinants that influence the frequency and specificity of misalignment-mediated frameshifts and deletions.     

DNA Sequences of Frameshift and Other Mutations Induced by Icr-170 in Yeast

ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G.C additions at sites containing monotonous runs of three G.C base pairs. However, some (Formula: see text) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G.C base pairs.     

Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain

We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers. While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site. Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis. Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations

The frequency and specificity of mutations produced in vitro by eucaryotic DNA polymerase-beta have been determined in a forward mutation assay using a 250-base target sequence in M13mp2 DNA. Homogeneous DNA polymerase-beta, isolated from four different sources, produces mutations at a frequency of 4-6%/single round of gap-filling DNA synthesis. DNA sequence analyses of 460 independent mutants resulting from this error-prone DNA synthesis demonstrate a wide variety of mutational events. Frameshift and base substitutions are made at approximately equal frequency and together comprise about 90% of all mutations. Two mutational "hot spots" for frameshift and base substitution mutations were observed. The characteristics of the mutations at these sites suggest that certain base substitution errors result from dislocation of template bases rather than from direct mispair formation by DNA polymerase-beta. When considering the entire target sequence, single-base frameshift mutations occur primarily in runs of identical bases, usually pyrimidines. The loss of a single base occurs 20-80 times more frequently than single-base additions and much more frequently than the loss of two or more bases. Base substitutions occur at many sites throughout the target, representing a wide spectrum of mispair formations. Averaged over a large number of phenotypically detectable sites, the base substitution error frequency is greater than one mistake for every 5000 bases polymerized. Large deletion mutations are also observed, at a frequency more than 10-fold over background, indicating that purified DNA polymerases alone are capable of producing such deletions. These data are discussed in relation to the physical and kinetic properties of the purified enzymes and with respect to the proposed role for this DNA polymerase in vivo.     

Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae.

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.     

Genetic effects of potassium dichromate in Schizosaccharomyces pombe.

In this review we outline the various factors which may contribute to the non-randomness of intragenic mutational spectra and the occurrence of hot spots. These factors include sample size limitation, particularly for sites of low mutability, and possible regions of low recombination potential. In addition, the nature of the gene product places great restraint on the detectability of either frameshift and premature chain-terminating mutations on one hand, or of the majority of missense mutations on the other. The nature of the Genetic Code itself also limits the mutational spectrum in so far as specific base pair substitutions lead only to a limited number of detectable amino acid replacements.Mutational hot spots may be a special example of the influence of neighbouring base pairs in the mutability of any given base pair. This is apparently true for frameshift mutations which tend to occur in runs of repeated base pairs or base pair doublets. Neigbouring base effects could operate not only at the level of initial reactivity with a mutagen, but also subsequently at the levels of DNA repair, recombination or replication. In some cases rare or modified bases may be responsible for neighbour effects. We suggest specific experimental approaches which seem likely to aid in the elucidation of these problems.     

The influence of DNA base ratios on intragenic mutational spectra in prokaryotes

Because of the degeneracy of the Genetic Code, a given amino acid sequence can be written in terms of either GC- or AT-rich DNA-specified mRNA codons. It is shown that the genetic consequences in terms of detectable single base pair substitution mutations are very different in such GC- or AT-rich sequences. Furthermore the occurrences of potential frameshift mutation hot-spots, in runs of repeated or reiterated base pairs, and sites of mutationally important base methylation, also differ markedly between AT- and GC-rich sequences. Thus the evolutionary pathways which can be followed by using single or multiple changes in the amino acid sequence of a given polypeptide will be very different in prokaryotes with GC-rich or AT-rich DNA.     

DNA sequence fine-structure analysis of ilvG (IlvG+) mutations of Escherichia coli K-12.

Six ilvG (IlvG+) mutations of Escherichia coli K-12 were transferred to recombinant plasmids, and the DNA sequence of each mutation was determined. This analysis confirmed that expression of the ilvG gene product (acetohydroxy acid synthase II) requires the deletion of a single base pair or the addition of two base pairs within ilvG to displace a frameshift site present in wild-type E. coli K-12. This system should be useful in the analysis of potential frameshift mutagens.     

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.     

Suppressible base substitution mutations induced by angelicin (isopsoralen) in the Escherichia coli lacI gene: implications for the mechanism of SOS mutagenesis.

Angelicin- plus near-UV-induced mutations were umuC dependent in Escherichia coli K-12. Angelicin, a monofunctional psoralen derivative, is believed to damage DNA almost exclusively at pyrimidine bases. To broaden our knowledge about the mutagenic specificity of SOS-dependent mutagens, we determined the mutational specificity of 233 suppressible lacI mutations induced by angelicin. More than 90% of the nonsense mutations arose via transversion substitutions. The three most frequently mutated sites were at A-T base pairs and accounted for more than one-third of all induced nonsense mutations. The two hottest sites were at the only occurrences of the 5'-TATA-3' tetranucleotide in lacI, a sequence expected to be a preferred binding site for a psoralen. Both A-T-to-T-A and A-T-to-C-G transversions were well induced by angelicin treatment, but the frequency of each transversion depended on the particular site. We also detected significant induction of transversion mutations at G-C sites. The induction of transversions by an SOS-dependent mutagen that generates lesions at pyrimidines supports the idea that DNA lesions influence the selection of bases that are incorporated via the process of SOS repair.     

Removal of Frameshift Intermediates by Mismatch Repair Proteins in Saccharomyces cerevisiae

Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting -1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a -1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates.     

Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride

In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.     

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.     

Nuclear magnetic resonance investigation of primer--template models: formation of a pyrimidine bulge upon misincorporation

Our previous studies have shown that misaligned structures can occur upon misincorporation of a dNTP opposite thymine templates. The formation of misaligned structures during DNA replication, if not repaired properly, can be bypassed and extended by low-fidelity polymerases and ultimately lead to mutations. In this study, the base pair structures at the replicating sites of a set of primer-template models which mimic the situation upon misincorporation of a dNTP opposite cytosine templates have been determined. High-resolution NMR structural results show that misaligned structures with a C-bulge can be formed upon incorporation of dCTP, dTTP, and dATP opposite 5'-GC, 5'-AC, and 5'-TC templates, respectively. The stabilities of misaligned structures depend on the types of terminal base pairs at the replicating sites. Together with the structural findings in thymine templates, we conclude that terminal G.C and C.G base pairs always contribute a larger stabilizing effect to the misaligned structures containing a pyrimidine bulge than terminal A.T and T.A base pairs. Misalignment and thus deletion mutation are more likely to occur if misincorporation of a nucleotide opposite a pyrimidine template can cause template slippage to form a terminal G.C or C.G base pair. Although misalignment also occurs when the newly formed terminal base pair is an A.T base pair or a T.A base pair, both misaligned and mismatched conformers coexist, which can lead to deletion and substitution mutations, respectively.     

Analysis of the role of the pseudoknot component in the SRV-1 gag-pro ribosomal frameshift signal: loop lengths and stability of the stem regions.

The simian retrovirus-1 (SRV-1) gag-pro frameshift signal was identified in previous work, and the overall structure of the pseudoknot involved was confirmed (ten Dam E, Brierley I, Inglis S, Pleij C, 1994, Nucleic Acids Res 22:2304-2310). Here we report on the importance of specific elements within the pseudoknot. Some mutations in stem S1 that maintain base pairing have reduced frameshift efficiencies. This indicates that base pairing in itself is not sufficient. In contrast, frameshifting correlates qualitatively with the calculated stability of mutations in S2. The stems thus play different roles in the frameshift event. The nature of the base in L1 has little influence on frameshift efficiency. It is however required to bridge S2; deleting it lowers frameshifting from 23 to 9%. In L2, frameshift efficiency was not affected in a mutant that changed 10 to 12 bases. This makes it unlikely that the primary sequence of L2 plays a role in -1 frameshifting, in contrast to readthrough in Moloney murine leukemia virus (Wills N, Gesteland R, Atkins J, 1994, EMBO J 13:4137-4144). Deletions of 2 and 3 bases gave more frameshifting than the wild type, probably reflecting the increased stability of the pseudoknot due to a shorter loop L2. Deleting even more bases reduces frameshifting compared to wild-type levels. At this point, stress will build up in L2, and this will reduce overall pseudoknot stability.     

To slip or skip, visualizing frameshift mutation dynamics for error-prone DNA polymerases

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) mu catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol mu and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol mu switches to dNTP-stabilized misalignment. pol beta generates -1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.     

Abasic frameshift in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations

We have determined the three-dimensional structure of a non-self-complementary oligodeoxynucleotide duplex that contains a model abasic site. The duplex contains six GC base pairs plus the abasic site at the center of one strand and corresponds to an abasic frameshift. Two-dimensional NMR studies on the nonexchangeable protons show that the guanine bases on either side of the abasic site are stacked over each other and that the abasic site is rotated out of the helix. Close proton-proton interactions are observed between the H4' proton of the abasic site and sugar protons of the guanosine in the 5' direction, which allows the position of the free sugar to be well-defined. NOE buildup curves from NOESY spectra recorded at very short mixing times were used to calculate a set of interproton distances. This data set was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two conformations that differ in the sugar conformation of the guanosine next to the abasic site in the 3' direction were necessary to fit all the NMR data. One of these two conformations could only be stabilized by addition of counterions at specific sites.