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.     

HSM2 (HMO1) gene participates in mutagenesis control in yeast Saccharomyces cerevisiae

We have previously reported about a new Saccharomyces cerevisiae mutation, hsm2-1, that results in increase of both spontaneous and UV-induced mutation frequencies but does not alter UV-sensitivity. Now HSM2 gene has been genetically and physically mapped and identified as a gene previously characterized as HMO1, a yeast homologue of human high mobility group genes HMG1/2. We found that hsm2 mutant is slightly deficient in plasmid-borne mismatch repair. We tested UV-induced mutagenesis in double mutants carrying hsm2-1 mutation and a mutation in a gene of principal damaged DNA repair pathways (rad2 and rev3) or in a mismatch repair gene (pms1 and recently characterized in our laboratory hsm3). The frequency of UV-induced mutations in hsm2 rev3 was not altered in comparison with single rev3 mutant. In contrast, the interaction of hsm2-1 with rad2 and pms1 was characterized by an increased frequency of UV-induced mutations in comparison with single rad2 and pms1 mutants. The UV-induced mutation frequency in double hsm2 hsm3 mutant was lower than in the single hsm2 and hsm3 mutants. The role of the HSM2 gene product in control of mutagenesis is discussed.     

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells.

A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia coli) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G . C----A . T transitions; the other transition (A . T----G . C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.     

Disruption of Xpg increases spontaneous mutation frequency, particularly A:T to C:G transversion.

Cells isolated from Xpg (the mouse counterpart of XPG)-disrupted mice underwent premature senescence and showed early onset of immortalization, suggesting that Xpg might be involved in genetic stability. Recent studies showed that human XPG, in addition to its function in the nucleotide excision repair (NER), was involved in the repair of oxidative base damages such as thymine glycol (Tg) and 8-oxo-guanine (8-oxoG), and this may explain the genetic instability observed in Xpg-deficient cells. To clarify this point, we determined spontaneous mutation frequencies and the type of spontaneous base substitution mutations in cells obtained from normal and Xpg-deficient mice using the supF shuttle vector (pNY200) for mutation assay. The spontaneous mutation frequency of the supF gene in pNY200 propagated in the Xpg-deficient cells was about three times higher than that in normal cells, indicating the importance of Xpg in reducing the frequency of spontaneous mutations. The frequency of spontaneous base substitution mutations at A:T sites, particularly that of the A:T to C:G transversion, increased markedly in the Xpg-deficient cells.     

Mutagenicity of tamoxifen DNA adducts in human endometrial cells and in silico prediction of p53 mutation hotspots

Tamoxifen elevates the risk of endometrial tumours in women and alpha-(N(2)-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with alpha-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N(2)-tamoxifen and dG-N(2)-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both alpha-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for alpha-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only alpha-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC-->TA transversions. Application of the LwPy53 algorithm to the alpha-acetoxytamoxifen spectrum predicted strong GC-->TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N(2)-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.     

Exonuclease-deficient polymerase mutant of herpes simplex virus type 1 induces altered spectra of mutations

The effect of exonuclease activity of the herpes simplex virus DNA polymerase (Pol) on DNA replication fidelity was examined by using the supF mutagenesis assay. The recombinants with exonuclease-deficient Pol, containing an integrated supF gene in the thymidine kinase locus (tk), exhibited supF mutation frequencies ranging from 0.14 to 5.6%, consistent with the tk mutation frequencies reported previously (Y. T. Hwang, B.-Y. Liu, D. M. Coen, and C. B. C. Hwang, J. Virol. 71:7791-7798, 1997). The increased mutation frequencies were 10- to 500-fold higher than those observed for wild-type Pol recombinants. The increased mutation frequencies also were significantly higher than those of supF mutant replicated by exonuclease-deficient Pols in the plasmid-borne assay. Furthermore, characterization of supF mutants demonstrated that recombinants with a defective exonuclease induced types and distributions of supF mutations different from those induced by wild-type Pol recombinants. The types of supF mutations induced by exonuclease-deficient recombinants differed between the plasmid- and genome-based assays. The spectra of supF mutations also differed between the two assays. In addition, exonuclease-defective viruses also induced different spectra of supF and tk mutations. Therefore, both the assay methods and the target genes used for mutagenesis studies can affect the repication fidelity of herpes simplex virus type 1 Pol with defective exonuclease activity.     

Molecular changes in UV-induced and γ-ray-induced mutations in human lymphoblastoid cells

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 γ-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by γ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.     

The role of DNA polymerase eta in UV mutational spectra

UV irradiation generates predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is encoded by the POLH (XPV) gene in humans. In order to clarify the specific role of Pol eta in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells. This strategy provides an advantage over studying mutagenesis in cell lines derived from normal individuals and XP-V patients, since the genetic background of the cells is identical. Synthetic RNA duplexes were used to inhibit Pol eta expression in 293T cells. The reduction of Pol eta mRNA and protein was greater than 90%. The supF shuttle vector was irradiated with UVC and replicated in 293T cells in presence of anti-Pol eta siRNA. The supF mutant frequency was increased by up to 3.6-fold in the siRNA knockdown cells relative to control cells confirming that Pol eta plays an important role in mutation avoidance and that the pol eta knockdown was efficient. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Surprisingly, neither the type of mutations nor their distribution along the supF gene were substantially different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. The data are compatible with two models. (i) Incorrect replication of cytosine-containing photoproducts by a polymerase other than Pol eta produces similar mutations as when Pol eta is present but at a higher frequency. (ii) Due to lack of Pol eta or low levels of remaining Pol eta, lesion replication is delayed allowing more time for cytosine deamination within CPDs to occur. We provide proof of principle that siRNA technology can be used to dissect the in vivo roles of lesion bypass DNA polymerases in DNA damage-induced mutagenesis.     

Test of models for the sequence specificity of UV-induced mutations in mammalian cells

We have used mathematical modeling and statistical analysis to examine the correlation between UV-induced DNA damage and resulting base-substitution mutations in mammalian cells. The frequency and site specificity of UV-induced photoproducts in the supF gene of the pZ189 shuttle vector plasmid were compared with the frequency and site specificity of base-substitution mutations induced upon passage of the UV-irradiated vector in monkey cells. The hypothesis that the observed mutational spectrum is due to a preferential insertion of adenosine opposite UV photoproducts in the DNA template was found to best explain the mutational data. Models in which it was postulated that only (6-4) photoproducts, and not cyclobutane dimers, are mutagenic, or that the relative frequency of photoproduct formation does not influence mutation frequencies, fit the data much less well. This analysis demonstrates that molecular mechanisms of mutagenesis in mammalian cells can be deduced from mutational data obtained with a shuttle vector system.     

Peroxyl radical mediated oxidative DNA base damage: implications for lipid peroxidation induced mutagenesis

Endogenous DNA damage induced by lipid peroxidation is believed to play a critical role in carcinogenesis. Lipid peroxidation generates free radical intermediates (primarily peroxyl radicals, ROO(*)) and electrophilic aldehydes as the principal genotoxicants. Although detailed information is available on the role of aldehyde base adducts in mutagenesis and carcinogenesis, the contribution of peroxyl radical mediated DNA base damage is less well understood. In the present study we have mapped oxidative base damage induced by peroxyl radicals in the supF tRNA gene and correlated this information with peroxidation-induced mutations in several human fibroblast cell lines. Nearly identical patterns of oxidative base damage were obtained from reaction of DNA with either peroxidizing arachidonic acid (20:4omega6) or peroxyl radicals generated by thermolysis of ABIP in the presence of oxygen. Oxidative base damage primarily occurred at G and C. Transversions at GC base pairs in the supF gene were the major base substitution detected in all cell lines. Peroxyl radical induced tandem mutations were also observed. Many mutation hot spots coincided with sites of mapped oxidative lesions, although in some cases hot spots occurred adjacent to the damaged base. Evidence is presented for the involvement of 8-oxodG in the oxidation of DNA by ROO(*). These results are used to interpret some key features of previously published mutation spectra induced by lipid peroxidation in human cells.     

The role of DNA polymerase iota in UV mutational spectra

UVB (280-320 nm) and UVC (200-280 nm) irradiation generate predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is a DNA damage-tolerant and error-prone DNA polymerase encoded by the POLH (XPV) gene in humans. Another member of the Y family of error-prone DNA polymerases is POLI encoding DNA polymerase iota (Pol iota). In order to clarify the specific role of Pol iota in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells, similar as we have previously done for Pol eta. Synthetic RNA duplexes were used to efficiently inhibit Pol iota expression in 293 T cells. The supF shuttle vector was irradiated with 254 nm UVC and replicated in 293 T cells in presence of anti-Pol iota siRNA. Surprisingly, there was a consistent reduction of recovered plasmid from cells with Pol iota knockdown and this was independent of UV irradiation of the plasmid. The supF mutant frequency was unchanged in the siRNA knockdown cells relative to control cells confirming that Pol iota does not play an important role in UV mutagenesis. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Neither the type of mutations nor their distribution along the supF gene were significantly different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. These results show that Pol iota has no significant role in UV lesion bypass and mutagenesis in vivo and provides some initial data suggesting that this polymerase may be involved in replication of extrachromosomal DNA.     

Computational analysis of mutation spectra

Mutation frequencies vary along a nucleotide sequence, and nucleotide positions with an exceptionally high mutation frequency are called hotspots. Mutation hotspots in DNA often reflect intrinsic properties of the mutation process, such as the specificity with which mutagens interact with nucleic acids and the sequence-specificity of DNA repair/replication enzymes. They might also reflect structural and functional features of target protein or RNA sequences in which they occur. The determinants of mutation frequency and specificity are complex and there are many analytical methods for their study. This paper discusses computational approaches to analysing mutation spectra (distribution of mutations along the target genes) that include many detectable (mutable) positions. The following methods are reviewed: mutation hotspot prediction; pairwise and multiple comparisons of mutation spectra; derivation of a consensus sequence; and analysis of correlation between nucleotide sequence features and mutation spectra. Spectra of spontaneous and induced mutations are used for illustration of the complexities and pitfalls of such analyses. In general, the DNA sequence context of mutation hotspots is a fingerprint of interactions between DNA and DNA repair/replication/modification enzymes, and the analysis of hotspot context provides evidence of such interactions.     

Mutation spectra in supF: approaches to elucidating sequence context effects

Shuttle vectors carrying the supF suppressor tRNA gene were originally developed for mutagenesis experiments in primate and human cells. Since then, the supF gene has been used as a mutation reporter in other mammalian cells, yeast, Escherichia coli, and transgenic mice. The widespread use of the vector for studies of many DNA reactive agents has produced a large database of mutation spectra. These provide primary information on the kinds and distribution of mutations provoked by many agents and, in many instances, allow comparisons between related agents or the same agent in different cell backgrounds. In this review we will discuss some of these data with a primary focus on the interpretation of UV mutation spectra. We will also describe our development and application of custom supF marker genes as an approach to studying the effect of sequence context on mutation hotspots and cold spots. Our studies suggest that C-C photoproducts are not mutagenic in certain sequence contexts in which T-C photoproducts are mutation hotspots. In addition, we have found several examples of sequence context effects acting as much as 80 bases away from the site of mutation. We will consider some of the problems raised by these studies and the possible resolution of some of them offered by the newly discovered family of damage bypass DNA polymerases.     

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.     

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.     

Deamination of 5-methylcytosines within cyclobutane pyrimidine dimers is an important component of UVB mutagenesis

UVB mutagenesis is characterized by an abundance of C --> T and 5-methylcytosine --> T transitions at dipyrimidine sequences. It is not known how these mutations might arise. One hypothesis is that UV-induced mutations occur only after deamination of the cytosine or 5-methylcytosine within the pyrimidine dimer. It is not clear how methylation of cytosines at the 5-position influences deamination and how this affects mutagenesis. We have now conducted experiments with a CpG-methylated supF shuttle vector that was irradiated with UVB and then incubated at 37 degrees C to allow time for deamination before passage through a human cell line to establish mutations. This led to a significantly increased frequency of CC --> TT mutations and of transition mutations at 5'-PymCG-3' sequences. A spectrum of deaminated cis-syn cyclobutane pyrimidine dimers in the supF gene was determined using the mismatch glycosylase activities of MBD4 protein in combination with ligation-mediated PCR. Methylation at the C-5 position promoted the deamination of cytosines within cis-syn cyclobutane pyrimidine dimers, and these two events combined led to a significantly increased frequency of UVB-induced transition mutations at 5'-PymCG-3' sequences. Under these conditions, the majority of all supF mutations were transition mutations at 5'-PymCG-3', and they clustered at several mutational hot spots. Exactly these types of mutations are frequently observed in the p53 gene of nonmelanoma skin tumors. This particular mutagenic pathway may become prevalent under conditions of inefficient DNA repair and slow proliferation of cells in the human epidermis.     

UV-induced replicating instability in Schizosaccharomyces pombe

A pigmented adenine-requiring strain of S. pombe has been used to study some aspects of the UV-induced replicating instabilities in this yeast. The effect of the repair capacity of a cell on the frequency of such instabilities has been investigated by using three different radiation-sensitive mutants and by studying the influence of caffeine on the frequency of secondary mosaics. Data showed that UV-induced replicating instabilities are not influenced by changes in the repair capacity of the treated cell. Since two of the sensitive mutants used are known to have high spontaneous mutation rates and the third one shows reduced UV-induced mutability, the induction of replicating instabilities differs in this respect both from the induction of spontaneous and UV-induced mutations.     

Replication fidelity of the supF gene integrated in the thymidine kinase locus of herpes simplex virus type 1

Recombinant viruses were constructed to have an Escherichia coli replicon containing a mutagenesis marker, the supF gene, integrated within the thymidine kinase locus (tk) of herpes simplex virus type 1. These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the supF gene. A mutation frequency of approximately 10(-4) was observed for wild-type strain KOS-derived recombinants in their replication of the supF gene. However, recombinants derived from the PAA(r)5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the tk gene, had three- to fourfold increases in supF mutation frequency (P < 0.01), a result similar to that exhibited when the supF gene was induced to replicate as episomal DNA (Y. T. Hwang, B.-Y. Liu, C.-Y. Hong, E. J. Shillitoe, and C. B. C. Hwang, J. Virol. 73:5326-5332, 1999). Thus, the PAA(r)5 Pol mutant had an antimutator function in replicating the tk gene and was less accurate in replicating the supF gene than was the wild-type strain. The spectra of mutations and distributions of substituted bases within the supF genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA. Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication. Furthermore, the replication mode of the target gene also affected the mutational spectrum.     

Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT gene.

DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.