Mutation spectrum of heat-induced abasic sites on a single-stranded shuttle vector replicated in mammalian cells.

The mutational potency of apurinic/apyrimidinic (AP) sites induced by heat-treatment under acidic conditions has been studied in mammalian cells. Abasic sites were induced on a single-stranded DNA shuttle vector carrying the supF tRNA gene, eliminating, therefore, any ambiguity concerning the damaged strand. This vector was able to replicate both in mammalian cells and in bacteria where the mutations induced in animal cells on the supF tRNA gene were screened by the white/blue beta-galactosidase assay in the presence of isopropyl-1-thio-beta-D-galactopyranoside and 5-bromo-4-chloro-3-indoyl-beta-D-galactoside. All white colonies contained plasmid with a mutation on the target gene which was directly sequenced. Our results show that one AP site was induced/22 min of heating as measured by sensitivity of DNA to alkali denaturation or treatment with the AP-endonuclease activity of the FPG protein (Fapy-DNA glycosylase). Putative AP sites decrease survival of the plasmid with a lethal hit of one AP site/single-stranded molecule. Mutation frequency was increased by a factor of approximately six after 2 h at 70 degrees C. Most of the induced mutations were point mutations not distributed at random and clustered in the gene region which will give rise to the mature tRNA. Mutations were abolished by treatments that eliminated AP sites such as alkali treatment or incubation with the Fapy-DNA glycosylase protein. Under our experimental conditions, when only single mutations were taken into account, the order of base insertion opposite AP sites was G greater than A greater than T greater than C.     

Mutation spectrum following transfection of ultraviolet-irradiated single-stranded or double-stranded shuttle vector DNA into monkey cells

We designed a shuttle vector system that allowed a comparison of the mutation spectrum on the supF target gene after transfection of single-stranded or double-stranded DNA into monkey cells. Single-strand-derived plasmids exhibited a spontaneous mutation frequency tenfold higher than double-strand-derived ones. These spontaneous mutations comprised deletions and point substitutions. This system was applied to the study of ultraviolet-induced mutagenesis. Single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after identical ultraviolet-irradiation. The use of single-stranded DNA allowed us to confirm and complete the data about the targeting of ultraviolet-induced mutations and the exact nature of the base changes involved. One class of mutations was more frequent after transfection of ultraviolet-irradiated single-stranded DNA than for double-stranded DNA: frameshifts represented 10% of the mutants. Multiple mutations, attributed by some authors to an error-prone excision repair process, have also been observed in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, although it cannot be a direct substrate for excision repair.     

Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.     

DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs.

A number of eukaryotic DNA binding proteins have been isolated by screening phage expression libraries with DNA probes containing the binding site of the DNA-binding protein. This methodology was employed here to isolate clones of the factor that interacts with the W box element of the human major histocompatibility complex HLA-DQB gene. Surprisingly, several cDNA clones of YB-1, a cDNA clone that was previously isolated with a CCAAT element-containing sequence were found. Independently, the screening of phage expression libraries with depurinated DNA resulted in the isolation of YB-1 and dbpA, a previously isolated cDNA that has homology to YB-1. Additional characterization of YB-1 showed that it bound a wide variety of DNA sequences and suggested that the binding of this protein is promiscuous. Furthermore, we show that both YB-1 and dbpA bind to depurinated DNA better than undamaged DNA and that the extent of specificity of binding is influenced by Mg2+. Due to the lack of sequence specificity and high degree of binding to depurinated DNA, we suggest that these proteins might be involved in chromosome functions such as maintenance of chromatin structure or DNA repair that do not require sequence-specific binding.     

Synthesis and properties of oligodeoxyribonucleotides containing abasic site.

Dodecanucleotide, d(CGCXCGCGTGCG), containing abasic site at desired position in the sequence was synthesized by solid-phase triester method. The introduced moiety (X) WAS N-(2'-deoxy-beta-D-erythropentofuranosyl) formamide.     

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.     

Mutagenic properties of a unique abasic site in mammalian cells

The mutagenic properties of a true unique abasic site located opposite a guanine residue were studied. An oligonucleotide containing a chemically-produced abasic site was inserted into a shuttle vector able to replicate both in simian cells and in bacteria. Plasmid DNA was rescued from simian cells and screened in bacteria by differential hybridization with a labelled oligonucleotide probe. Mutations were easily detected and sequenced. Results showed that opposite a guanine the abasic site was error free repaired or replicated by mammalian cells with an efficiency of 99%. Point mutations occurred at a frequency of approximately 1% in control host cells and at more than 3% in UV-pre-irradiated host cells. Adenine, cytosine or thymine were found to have been inserted opposite the abasic site. No preferential insertion for a particular base was observed in contrast to that reported in bacteria.     

Mutagenic spectrum resulting from DNA damage by oxygen radicals.

Oxygen free radicals are highly reactive species that damage DNA and cause mutations. We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe2+. The Fe2(+)-treated DNA was transfected into component Escherichia coli, and mutants within the nonessential lac Z alpha gene for beta-galactosidase were identified by decreased alpha-complementation. The frequency of mutants obtained with 10 microM Fe2+ was 20- to 80-fold greater than that obtained with untreated DNA. Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS "error-prone" response. The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals. The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions. Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G----C transversions, followed by C----T transitions and G----T transversions. The clustering of mutations at distinct gene positions suggests that Fe2+/oxygen damage to DNA is nonrandom. This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.     

Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking poison sequences and containing 16 cloning sites, has been the most satisfactory.     

Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo.

In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro.     

Spontaneous and ultraviolet-induced mutations on a single-stranded shuttle vector transfected into monkey cells.

The shuttle vector plasmid PCF3A, carrying the supF target gene, can be transfected into monkey COS7 cells as single-stranded or double-stranded DNA. Single strand-derived plasmid progeny exhibited a 10-fold higher spontaneous mutation frequency than double strand-derived progeny. The location of spontaneous mutations obtained after transfection of the single-stranded vector shared similarities with that for double-stranded vectors. However, the nature of base changes was very different. Single-stranded PCF3A DNA was used to study ultraviolet-induced mutagenesis. An earlier report (Madzak and Sarasin, J. Mol. Biol., 218 (1991) 667-673) showed that single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after ultraviolet irradiation. In the present report, sequence analysis of mutant plasmids is presented. The use of a single-stranded vector allowed us to show the targeting of mutations at putative lesion sites and to determine the exact nature of the base implicated in each mutation. Frameshift mutations were more frequent after transfection of control or irradiated plasmid as single-stranded DNA than as double-stranded DNA. Multiple mutations, observed at a high frequency in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, could be due to an error-prone polymerisation step acting on a single-stranded template.     

Analysis of Aspergillus niger transformants for single site integration and vector recombination

Summary In order to understand the mechanism of integrative transformation, bulk DNA from several transformants of Aspergillus niger was assaved for the presence of the heterologous vector p3SR2. In all cases vector sequences and recombinant vector fragments were found. Transformant T31-6 containing seven plasmid-probed bands was studied in detail. Recloning of six of these plasmids in Escherichia coli revealed that internal recombination events had produced deletions and insertions. Only one plasmid was isolated among 402 analysed clones that had involved A. niger DNA. Sequence analysis revealed that the integration of vector p3SR2 occurred via its A. nidulans amdS ⁺ sequence. These data support the assumption that the integration of multiple copies of p3SR2 occurred at a single genomic site.Offprint requests to: K. Esser     

An abasic site analogue activates a c-Ha-ras gene by a point mutation at modified and adjacent positions.

Synthetic c-Ha-ras genes with an analogue of an abasic site in the first or the second position of codon 12, or in the second position of codon 61 were constructed and transfected into NIH3T3 cells. The genes with the lesions in codon 12 exhibited more focus formation than a normal c-Ha-ras gene, while the gene with the lesion in codon 61 did not. Transformed cells were isolated from the foci, and the c-Ha-ras genes present in the transformants were analysed. A point mutation to A in the modified position was found most frequently in the cases of ras genes modified in codon 12. Surprisingly, point mutations in the adjacent position were also detected. These results indicate that dTMP, and not dAMP, was mainly incorporated into the sites opposite to the abasic site analogue, and that incorrect deoxynucleotides were incorporated in the position adjacent to the abasic site analogue.     

Mutation of a single conserved nucleotide between the cloverleaf and internal ribosome entry site attenuates poliovirus neurovirulence

The chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles yielded virus whose mouse neurovirulence was highly attenuated (J. Cello, A. V. Paul, and E. Wimmer, Science 297:1016-1018, 2002). Compared to the wild-type PV1 (Mahoney) [PV1(M)] sequence, the synthetic virus genome harbored 27 nucleotide (nt) changes deliberately introduced as genetic markers. Of the 27 nucleotide substitutions, the UA-to-GG exchanges at nucleotides 102/103, mapping to a region between the cloverleaf and the internal ribosome entry site (IRES) in the 5'-nontranslated region, were found to be involved in the observed attenuation phenotype in mice. The UA/GG mutation at nt 102/103 in the synthetic PV1(M) [sPV1(M)] background conferred also a ts phenotype of replication to the virus in human neuroblastoma cells. Conversely, the exchange of GG to wild-type (wt) UA at 102/103 in an sPV1(M) background restored wt neurovirulence in CD155 transgenic (tg) mice and suppressed the ts phenotype in SK-N-MC cells. All poliovirus variants replicated well in HeLa cells at the two temperatures, regardless of the sequence at the 102/103 locus. Analyses of variants isolated from sPV(M)-infected CD155 tg mice revealed that the G(102)G(103)-to-G(102)A(103) reversion alone reestablished the neurovirulent phenotype. This suggests that a single mutation is responsible for the observed change of the neurovirulence phenotype. sPV1(M) RNA is translated in cell extracts of SK-N-MC cells with significantly lower efficiency than PV1(M) RNA or sPV1(M) RNA with a G(102)-to-A(102) reversion. These studies suggest a function for the conserved nucleotide (A(103)) located between the cloverleaf and the IRES which is important for replication of PV in the central nervous system of CD155 tg mice and in human cells of neuronal origin.     

Gain or loss of diabetogenicity resulting from a single point mutation in recombinant encephalomyocarditis virus.

Molecular pathogenic mechanisms for virus-induced disease have received considerable attention. Encephalomyocarditis (EMC) virus-induced diabetes in mice has been extensively studied to elucidate the cellular and molecular mechanisms involved in the development of this disease. In this study, we report for the first time that a single point mutation at nucleotide position 3155 or 3156 of the recombinant EMC viral genome, located on the major capsid protein VP1, which causes an amino acid change, results in the gain or loss of viral diabetogenicity. A G base at nucleotide position 3155 (alanine at amino acid position 776 of the EMC virus polyprotein [Ala776]; GCC) results in viral diabetogenicity, whereas the substitution of other bases at the same or next position results in a loss of viral diabetogenicity. This finding provides clear evidence that a point mutation at a critical site in a viral genome affects the ability of the virus to cause a cell-specific disease.     

The genealogy, site frequency spectrum and ages of two nested mutant alleles

In this paper we consider the genealogy of two nested mutant alleles, assuming the constant-size neutral coalescent model with infinite sites mutation. We study the conditional genealogy and derive explicit formulas for the joint and marginal site frequency spectra for the double, single and zero mutant allele. In addition, we find the mean ages of the two mutations. We show that the age of the youngest mutation does not depend on the frequency of the single mutant allele and that the frequency spectra for the single mutant allele and the zero mutant allele are the same.     

A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities.

Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis. A major product of free radical DNA damage is the formation of 8-oxoguanine, which is a highly mutagenic base modification produced by oxidative stress. Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8-oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction. The significance of this DNA repair activity acting on 8-oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8-oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-->T transversions. The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites. These results raise the possibility that DNA repair may be associated with protein translation.     

An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations

We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.