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PubMed1997

An Escherichia coli topB mutant increases deletion and frameshift mutations in the supF target gene.

We have improved a system to examine forward mutations that occurred in the supF gene of Escherichia coli placed on a multicopy plasmid. Using this system, we determined the mutational specificity for a topB deletion mutator in which topoisomerase III is hampered. The frequency of supF- mutations in topB strain was 4.9 x 10(-7), that is essentially the same as that in wild-type strain, 3.1 x 10(-7). Half the number of the supF- mutations were large deletions, where a specific deletion among a 10-base pair direct repeat dominated, but other types of deletions were also found. Most of the deletions were associated with the presence of directly repeated sequences capable of accounting for their endpoints. Frameshift mutations in topB strain also significantly increased compared with those of wild-type (17 vs. 2%). Base substitutions comprised 27% of the events, specificity of which in topB strain was the same as that in wild-type strain. The present data suggest that topB is a novel class of mutator that strongly induces repeated sequence dependent deletion mutagenesis and high frequencies of frameshift mutagenesis.

Base Sequence↗
PubMed2001

Induction of -2 frameshift mutations by 2-nitrofluorene, N-hydroxyacetylaminofluorene, and N-2-acetylaminofluorene in reversion assays in Escherichia coli strains differing in permeability and acetyltransferase activity.

The mutagenicity of 2-nitrofluorene (NF), N-hydroxyacetylaminofluorene (N-OH-AAF), and N-2-acetylaminofluorene (AAF) was measured in strains of Escherichia coli that contain a lacZ allele that reverts by -2 frameshift mutations from CG(5) to CG(4). Mutagenesis was compared in a strain having wild-type permeability and metabolism, a strain with increased permeability caused by a lipopolysaccharide-defective (LPS(d)) mutation, a strain with N- and O-acetyltransferase (NAT/OAT) activity conferred by the Salmonella nat gene on plasmid pYG219, and a strain carrying both an LPS(d) mutation and pYG219. The LPS(d) mutation facilitated the measurement of mutagenicity but was not absolutely required, in that lower levels of mutagenicity were detected in LPS(+) strains. The NAT/OAT activity conferred by pYG219 strongly potentiated the mutagenicity of NF and N-OH-AAF. Surprisingly, AAF was mutagenic in the NAT/OAT LPS(d) strain without an exogenous P450 metabolic activation system. Its activity may be ascribable to the detection of a directly mutagenic impurity by the highly sensitive strain or to a low level of metabolic activation by the bacteria under the assay conditions. The findings add to the evidence that the lacZ allele derived from E. coli strain CC109 is an effective indicator of -2 frameshift mutagenesis and that strains expressing high levels of NAT/OAT activity are highly sensitive in monitoring the mutagenicity of nitroarenes and aromatic amides.

2-Acetylaminofluorene↗
PubMed1999

Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli.

To investigate the mutation spectrum of a well-known mutagen, methylglyoxal, and the influence of nucleotide excision repair (NER) on methylglyoxal-induced mutations, we treated wild-type and NER-deficient (uvrA or uvrC) Escherichia coli strains with methylglyoxal, and analyzed mutations in the chromosomal lacI gene. In the three strains, the cell death and the mutation frequency increased according to the dose of methylglyoxal added to the culture medium. The frequencies of methylglyoxal-induced base-pair substitutions were higher in the NER-deficient strains than in the wild-type strain, in the presence and absence of mucAB gene. Paradoxically, the frequency of methylglyoxal-induced TGGC frameshifts was higher in the wild-type strain than in the NER-deficient strains. When the methylglyoxal-induced mutation spectra in the presence and absence of mucAB gene are compared, the ratios of base-pair substitutions to frameshifts were increased by the effects of mucAB gene. In the three strains, more than 75% of the base-pair substitutions occurred at G:C sites, independent of the mucAB gene. When the mucAB gene was present, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. When the mucAB gene was absent, the predominant mutations differed in the three strains: in the wild-type and uvrC strains, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions, while in the uvrA strains, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. These results suggest that NER may be involved in both the repair and the fixation of methylglyoxal-induced mutations.

Base Pair Mismatch↗
PubMed1999

Frameshift mutation, microsatellites and mismatch repair.

The structure of eukaryotic DNA, with its repeated sequences, makes base addition and loss a major obstacle to the maintenance of genetic stability. As compared to the bacteria, much of the mismatch repair capacity of the eukaryotic cell must be devoted to the surveillance of frameshift changes. Any alteration in the activity of proteins which recognize frameshifts or which hold the DNA in place during replication is likely to result in genomic instability.

Base Pair Mismatch↗
PubMed2003

Different frameshift mutation spectra in non-repetitive DNA of MutSalpha- and MutLalpha-deficient fission yeast cells.

A frameshift reversion assay has been established for Schizosaccharomyces pombe, which allows detection of deletions and insertions of nucleotides in a non-repetitive DNA sequence. Compared to wild type, frameshift mutation rates were increased in the mismatch repair (MMR) mutants msh2, msh6, mlh1, and pms1, but not in a swi4 strain (defective in the Msh3 homologue). Rates were also elevated in the DNA nuclease-deficient strains rad2 (defective in the FEN-1 homologue) and exo1. In MutSalpha-deficient strains, msh2 and msh6, most of the reversions were 1bp deletions. In contrast, mlh1 and pms1 mutants, defective in MutLalpha, accumulated significantly more 2bp insertions, preferentially of the type CG to (CG)(2). Such duplications were less frequent in double mutants additionally defective in msh2, msh6, rad2, or exo1. Thus, accumulation of (CG)(2) in MutLalpha-deficient strains depends on the presence of MutSalpha, Rad2 and Exo1.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus.

The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G x U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CdeltaA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (+/-0.2) kcal mol(-1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (deltaG(0)37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability deltadeltaH = 6.6 (+/-2.1) kcal mol(-1) with deltadeltaG(0)37 comparable to that predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G x U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)6(3+) ions stabilize the A14 and deltaA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus deltaA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617).

Adenosine↗
PubMed1994

DNA sequence determinants of carcinogen-induced frameshift mutagenesis.

In order to examine the mechanisms of induced frameshift mutagenesis, we constructed double-stranded DNA plasmids which contain single N-2-acetylaminofluorene (AAF) adducts at specified positions within a run of contiguous guanine residues. The length of the homopolymeric run and the nature of the bases flanking the contiguous sequence were systematically varied. Monomodified plasmids were introduced into SOS-induced Escherichia coli, and -1 frameshift mutations were scored by means of a phenotypic assay. A strong positional effect of the DNA adduct within the contiguous sequence was observed irregardless of the nature of the flanking bases: the AAF-induced mutation frequency was 20-200-fold higher at the 3'-end of the contiguous sequence than at the 5'-end. In addition, for a given number of guanine residues flanking the GAAF adduct on its 5'-side, differences (up to 5-fold) in the induced mutation frequency were observed as a function of the base 3' to the adduct (CGGGAAFAT approximately CGGGAAFGT > CGGGAAFCT > CGGGAAFTT). These results are discussed, within the frame of an incorporation slippage model, in terms of differences in stability and occurrence of the slipped mutagenic intermediates.

2-Acetylaminofluorene↗
PubMed2005

Mechanism for N-acetyl-2-aminofluorene-induced frameshift mutagenesis by Escherichia coli DNA polymerase I (Klenow fragment).

N-Acetyl-2-aminofluorene (AAF) is a chemical carcinogen that reacts with guanines at the C8 position in DNA to form a structure that interferes with DNA replication. In bacteria, the NarI restriction enzyme recognition sequence (G1G2CG3CC) is a very strong mutational hot spot when an AAF adduct is positioned at G3 of this sequence, causing predominantly a -2 frameshift GC dinucleotide deletion mutation. In this study, templates were constructed that contained an AAF adduct at this position, and primers of different lengths were prepared such that the primer ended one nucleotide before or opposite or one nucleotide after the adduct site. Primer extension and gel shift binding assays were used to study the mechanism of bypass by the Escherichia coli DNA polymerase I (Klenow fragment) in the presence of these templates. Primer extension in the presence of all four dNTPs produced a fully extended product using the unmodified template, while with the AAF-modified template synthesis initially stalled at the adduct site and subsequent synthesis resulted in a product that contained the GC dinucleotide deletion. Extension product and gel shift binding analyses were consistent with the formation of a two-nucleotide bulge structure upstream of the active site of the polymerase after a nucleotide is incorporated across from the adduct. These data support a model in which the AAF adduct in the NarI sequence specifically induces a structure upstream of the polymerase active site that leads to the GC frameshift mutation and that it is this structure that allows synthesis past the adduct to occur.

2-Acetylaminofluorene↗
PubMed1998

Solution structure of N-(2-deoxy-D-erythro-pentofuranosyl)urea frameshifts, one intrahelical and the other extrahelical, by nuclear magnetic resonance and molecular dynamics.

The presence of a N-(2-deoxy-D-erythro pentofuranosyl)urea (henceforth referred to as deoxyribosylurea) residue, ring fragmentation product of a thymine, in a frameshift situation in the sequence 5'd(AGGACCACG).d(CGTGGurTCCT) has been studied by 1H and 31P nuclear magnetic resonance and molecular dynamics. At equilibrium, two species are found in slow exchange. We observe that the deoxyribosylurea residue can be either intra- or extrahelical within structures which otherwise do not deviate strongly from that of a B-DNA as observed by NMR. Our study suggests that this is determined by the nature and number of hydrogen bonds which this residue can form as a function of two possible isomers. There are two possible structures for the urea side chain, either cis or trans for the urido bond which significantly changes the hydrogen bonding geometry of the residue. In the intrahelical species, the cis isomer can form two good hydrogen bonds with the bases on the opposite strand in the intrahelical species, A4 and C5, which is not the case for the trans isomer. This results in a kink in the helical axis. For the major extrahelical species, the situation is reversed. The trans isomer is able to form two good hydrogen bonds, with G13 on the same strand and A7 on the opposite strand. For the extrahelical species, the cis isomer can form only one hydrogen bond. In this major structure the NMR data show that the bases which are on either side of the deoxyribosylurea residue in the sequence, G14 and T16, are stacked over each other in a way similar to a normal B-DNA structure. This requires the formation of a loop for the backbone between these two residues. This loop can belong to one of two families, right- or left-handed. In a previous study of an abasic frameshift [Cuniasse et al. (1989) Biochemistry 28, 2018-2026], a left-handed loop was observed, whereas in this study a right-handed loop is found for the first time in solution. The deoxyribosylurea residue lies in the minor groove and can form both an intra- and an interstrand hydrogen bond.

Computer Simulation↗
PubMed1999

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis.

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.

2-Aminopurine↗
PubMed2003

Identification of an HLA-A0201-restricted CTL epitope generated by a tumor-specific frameshift mutation in a coding microsatellite of the OGT gene.

Deficient DNA mismatch repair results in microsatellite instability and might induce shifts of translational reading frames of genes encompassing coding microsatellites. These may be translated in truncated proteins, including neo-peptide tails functioning as tumor rejection antigens, when presented in the context of MHC class I. Recently, others and we identified a frameshift mutation in the coding T(10) microsatellite of the O-linked N-acetylglucosamine transferase gene (OGT) occuring in up to 41% of microsatellite unstable colorectal cancers. Here we describe a novel HLA-A0201-restricted cytotoxic T lymphocyte (CTL)-epitope (28-SLYKFSPFPL; FSP06) derived from this mutant OGT-protein. FSP06-specific CTL-clones killed peptide-sensitized target cells and tumor cell lines expressing both HLA-A0201 and mutant OGT proteins. This demonstrates that FSP06 is endogenously expressed and represents a CD8(+)-T cell epitope. Our data corroborate the concept of frameshift peptides constituting a novel subset of tumor-associated antigens specifically encountered in cancer cells with deficient mismatch repair.

Amino Acid Sequence↗
PubMed2001

Identification of MICA alleles with a long Leu-repeat in the transmembrane region and no cytoplasmic tail due to a frameshift-deletion in exon 4.

MHC class I chain-related gene A (MICA) is located close to HLA-B gene and expressed in epithelial cells. The MICA gene is reported to be highly polymorphic as are the classical class I genes. To further assess the polymorphism in the MICA gene, we analyzed a total of 60 HLA-homozygous cells for the sequences spanning exons 2-6. In the analysis, four new MICA alleles were identified and six variations were recognized in exon 6. MICA*017, which was identified in three HLA-B57 homozygous cells (DBB, DEM and WIN), differed from MICA*002 in exon 3 and had a guanine deletion at the 3' end of exon 4. MICA*015 identified in an HLA-B45 homozygous cell (OMW) also had the same deletion that causes a frameshift mutation resulting in complete change of the transmembrane region and premature termination in the cytoplasmic tail; these alleles have a long hydrophobic leucine-rich region instead of the alanine repeat in the transmembrane region and terminate at the second position in the cytoplasmic domain. The frameshift deletion was found only in HLA-B45- or -B57-positive panels tested, suggesting a strong linkage disequilibrium between the deletion and B45 or B57. MICA*048, which was different in exon 5 from MICA*008, was identified in an HLA-B61 homozygous cell (TA21), while MICA*00901 identified in HLA-B51 homozygous cells (LUY and KT2) was distinguished from MICA*009 by exon 6.

Base Sequence↗
PubMed1999

Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot.

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

Crystallography, X-Ray↗
PubMed1997

GLI3 frameshift mutations cause autosomal dominant Pallister-Hall syndrome.

Pallister-Hall syndrome (PHS, M146510) was first described in 1980 in six newborns. It is a pleiotropic disorder of human development that comprises hypothalamic hamartoma, central polydactyly, and other malformations. This disorder is inherited as an autosomal dominant trait and has been mapped to 7p13 (S. Kang et al. Autosomal dominant Pallister-Hall syndrome maps to 7p13. Am. J. Hum. Genet. 59, A81 (1996)), co-localizing the PHS locus and the GLI3 zinc finger transcription factor gene. Large deletions or translocations resulting in haploinsufficiency of the GLI3 gene have been associated with Greig cephalopolysyndactyly syndrome (GCPS; M175700) although no mutations have been identified in GCPS patients with normal karyotypes. Both PHS and GCPS have polysyndactyly, abnormal craniofacial features and are inherited in an autosomal dominant pattern, but they are clinically distinct. The polydactyly of GCPS is commonly preaxial and that of PHS is typically central or postaxial. No reported cases of GCPS have hypothalamic hamartoma and PHS does not cause hypertelorism or broadening of the nasal root or forehead. The co-localization of the loci for PHS and GCPS led us to investigate GLI3 as a candidate gene for PHS. Herein we report two PHS families with frameshift mutations in GLI3 that are 3' of the zinc finger-encoding domains, including one family with a de novo mutation. These data implicate mutations in GLI3 as the cause of autosomal dominant PHS, and suggest that frameshift mutations of the GLI3 transcription factor gene can alter the development of multiple organ systems in vertebrates.

Abnormalities, Multiple↗
PubMed1998

BAX frameshift mutations in cell lines derived from human haemopoietic malignancies are associated with resistance to apoptosis and microsatellite instability.

Bax suppresses tumorigenesis in a mouse model system and Bax-deficient mice exhibit lymphoid hyperplasia suggesting that BAX functions as a tumour suppressor in human haemopoietic cells. We examined BAX expression in 20 cell lines derived from human haemopoietic malignancies and consistent with a potential tumour suppressor function, identified two cell lines, DG75 (a Burkitt lymphoma cell line) and Jurkat (a T-cell leukaemia line), which lacked detectable BAX expression. Apoptosis of DG75 cells induced by low serum or ionomycin was significantly delayed relative to similar Burkitt lymphoma cell lines with normal BAX levels. Although DG75 and Jurkat cells expressed several BAX RNA species including the prototypical BAX alpha RNA, the absence of BAX protein was due to single base deletions and additions in a polyguanine tract within the BAX open reading frame. These frameshift mutations result in premature termination of translation and have recently also been identified in some colon cancers with microsatellite instability. Although mismatch repair defects are not considered a common feature of haemopoietic malignancies, DG75 and Jurkat cells had widespread microsatellite instability and did not express detectable levels of MSH2. In Jurkat cells, lack of MSH2 expression was due to a point mutation in exon 13 of MSH2 resulting in premature termination of translation. Our results suggest that a pathway linking mismatch repair defects, BAX tumour suppressor frameshift mutations and resistance to apoptosis may be a key feature of some lymphomas and leukaemias.

Apoptosis↗
PubMed1999

Somatic frameshift mutations in the MBD4 gene of sporadic colon cancers with mismatch repair deficiency.

Defects of mismatch repair are thought to be responsible for carcinogenesis in hereditary non-polyposis colorectal cancer and about 15% of sporadic colon cancers. The phenotype is seen as microsatellite instability and is known to be caused either by mutations in mismatch repair genes or by aberrant methylation of these genes stabilizing their downregulation. Lack of repair of microsatellite sequence errors, created during replication, leads to a mutation-prone phenotype. Where mutations occur within mononucleotide tracts within exons they cause translation frameshifts, premature cessation of translation and abnormal protein expression. Such mutations have been observed in the TGFbetaRII, BAX, IGFIIR, MSH3 and MSH6 genes in colon and other cancers. We describe here frameshift mutations affecting the gene for the methyl-CpG binding thymine glycosylase, MBD4, in over 40% of microsatellite unstable sporadic colon cancers. The mutations all appear heterozygous but their location would ensure truncation of the protein between the methyl-CpG binding and glycosylase domains, thus potentially generating a dominant negative effect. It is thus possible that such mutations enhance mutation frequency at other sites in these tumours. A suggestion has been made that MBD4 (MED1) mutations may lead to an increased rate of microsatellite instability but this mechanism appears unlikely due to the nature of mutations we have found.

Base Pair Mismatch↗
PubMed2005

Caspase-8 gene is frequently inactivated by the frameshift somatic mutation 1225_1226delTG in hepatocellular carcinomas.

Evidence exists that alterations of the genes encoding apoptosis-related proteins contribute to either development or progression of human cancers. Caspase-8 plays a crucial role in the initiation phase of apoptosis. To explore the possibility that the genetic alteration of caspase-8 gene is involved in the development of hepatocellular carcinomas (HCCs), we have analysed the entire coding region of human caspase-8 gene for the detection of somatic mutations by polymerase chain reaction-single-strand conformation polymorphism in 69 HCCs with low-grade dysplastic nodule (LGDN, n=2) or high-grade dysplastic nodule (HGDN, n=2) or without any dysplastic nodules (n=65). Overall, we detected a total of nine somatic mutations in 69 HCCs (13.0%). Interestingly, all of the nine mutations were an identical frameshift mutation with two base-pair deletion (1225_1226delTG), which would result in a premature termination of amino-acid synthesis in the p10 protease subunit. In a patient sample, we detected the 1225_1226delTG mutation both in HCC and LDGN lesions, suggesting that caspase-8 mutation could be involved in the early stage of HCC carcinogenesis. We expressed the tumor-derived caspase-8 mutant in the cells and found that the mutant abolished cell death activity of caspase-8. Our data indicate that caspase-8 gene is frequently mutated in HCC and the majority of the mutations may be the frameshift mutation 1225_1226delTG. Also, the data suggest that caspase-8 gene mutation might lead to the loss of its cell death function and contribute to the pathogenesis of HCC.

Adaptor Proteins, Signal Transducing↗
PubMed1997

First description of a frameshift mutation in the alpha1-globin gene associated with alpha-thalassaemia.

A frameshift mutation in the alpha1-globin gene, responsible for a clinically mild alpha-thalassaemia phenotype, has been characterized in a Spanish woman. After excluding the most common forms of alpha-thalassaemia found in the Mediterranean area, both alpha-globin genes (alpha1 and alpha2) were amplified and analysed selectively by non-radioactive single-strand conformation polymorphism (SSCP). An abnormal SSCP mobility was present in the second exon of the alpha1-globin gene and direct sequence analysis revealed a 13 bp deletion (between codons 51 and 55) affecting a single allele. The consequence of this mutation is a reading frameshift leading to a novel amino acid coding sequence from codons 51-61 and a premature stop signal at new position 62, which results in a net reduction of the affected alpha-globin chain output. The presence of this new mutation was confirmed by restriction enzyme analysis of the specific PCR product.

Aged↗