Frameshift mutagenesis of lambda prophage by 9-aminoacridine, proflavin and ICR-191

Summary The changes in DNA base sequence induced in the lambda cI gene in an E. coli lysogen have been determined following mutagenesis by three acridine derivatives: 9-aminoacridine and proflavin, which bind reversibly to DNA; and ICR-191, which attaches covalently to DNA through a half-mustard group. For all three derivatives, most mutations are +1 and-1 frameshifts in runs of adjacent G:C pairs. The specificity of mutagenesis at various sites is similar for all three compounds. Prophage in mutL host cells, deficient in mismatch repair, are much more susceptible to mutagenesis by 9-aminoacridine. The induced mutations are also frameshifts, and the site specificity is the same as in lysogens of wild type cells. Thus, additions or deletions of single bases can be corrected by the mismatch repair system, but mismatch repair does not play an important role in determining the sequence specificity of the mutational events.     

Mutagenicity induced by UVC in Escherichia coli cells: reactive oxygen species involvement

We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen ((1)O(2)) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an (1)O(2) chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that (1)O(2) plays a predominant role in UVC-induced mutagenesis. (1)O(2) generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC-TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC-TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC-TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.     

Spontaneous and mutagen-induced loss of DNA mismatch repair in Msh2-heterozygous mammalian cells

We have developed a simple procedure that enables the efficient selection of cells that are deficient for DNA mismatch repair (MMR). This selection procedure was used to investigate the frequency of fortuitous MMR-deficient cells in a mouse embryonic stem cell line, heterozygous for the MMR gene Msh2. We found a surprisingly high frequency (3 x 10(-4)) of Msh2-deficient cells. The wild type Msh2 allele was almost invariably lost by loss of heterozygosity. Single treatments with the genotoxic agents ethylnitrosourea, UVC light and mitomycin C resulted in a further increase of the number of Msh2-/- cells in the heterozygous cell line. This increase was not only due to induced loss of the wild type allele but also to a selective growth advantage of preexisting Msh2-/- cells to ethylnitrosourea and UVC. Mitomycin C, in contrast to ethylnitrosourea and UVC, uniquely induced loss of heterozygosity at Msh2. These mechanistically different ways of loss of the wild type Msh2 allele reflect the different repair pathways processing these damages. Heterozygous germ line defects in one of the MMR genes underlie the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. Based on the results described here we hypothesize that mutagen-induced loss of MMR in the intestine of these patients contributes to the tissue specificity of carcinogenesis in HNPCC patients.     

Photoreactivation implicates cyclobutane dimers as the major promutagenic UVB lesions in yeast.

Previously we compared the mutational specificities of polychromatic UVB (285-320 nm) and UVC (254 nm) light in the SUP4-o gene of the yeast Saccharomyces cerevisiae. Striking similarities in the types and distributions of induced SUP4-o mutations were consistent with roles for cyclobutane dimers and pyrimidine(6-4)pyrimidone photoproducts in mutation induction by UVB. To assess the relative importance of cyclobutane dimers, we have now examined the effect of photoreactivation (PR), which specifically reverses these lesions, on UVB and UVC induction of SUP4-o mutations. PR reduced the frequencies of both UVB and UVC mutagenesis by approximately 75%. Collections of 138 and 158 SUP4-o mutants induced by treatment with UVB plus PR or UVC plus PR, respectively, were characterized by DNA sequencing and the results were compared to those for 208 UVB and 211 UVC-induced mutants analyzed earlier. PR decreased the frequency of UVB-induced G.C----A.T transitions by 85%, diminished the substitution frequencies at individual sites by 64% on average, and reduced the mutation frequencies at the five UVB hotspots by 87%. A more detailed examination revealed that the transition frequencies at the 3' base of 5'-TC-3' and 5'-CC-3' sequences were decreased by 90% and 72%, respectively. Finally, PR appeared to occur to the same extent on both the transcribed and non-transcribed strands of SUP4-o. Similar results were obtained for PR following UVC irradiation. Our findings indicate that cyclobutane dimers are responsible for the majority of UVB mutagenesis in yeast.     

Role of Purα in the cellular response to ultraviolet-C radiation

Pura is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Pura in Ultraviolet-C (UVC) radiation-induced DNA damage and nucleotide excision repair (NER). Mouse embryo fibroblasts from PURA-/- knockout mice, which lack Pura, showed enhanced sensitivity to UVC irradiation as assessed by assays for cell viability and clonogenicity compared to Pura positive control cultures. In reporter plasmid reactivation assays to measure the removal of DNA adducts induced in vitro by UVC, the Pura-negative cells were less efficient in DNA damage repair. Pura-negative cells were also more sensitive to UVC-induced DNA damage measured by Comet assay and showed a decreased ability to remove UVC-induced cyclobutane pyrimidine dimers. In wild-type mouse fibroblasts, expression of Pura is induced following S-phase checkpoint activation by UVC in a similar manner to the NER factor TFIIH. Moreover, co-immunoprecipitation experiments showed that Pura physically associates with TFIIH. Thus, Pura has a role in NER and the repair of UVC-induced DNA damage.     

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.     

Role of Purα in the cellular response to ultraviolet-C radiation

Purα is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Purα in Ultraviolet-C (UVC) radiation-induced DNA damage and nucleotide excision repair (NER). Mouse embryo fibroblasts from PURA^-/- knockout mice, which lack Purα, showed enhanced sensitivity to UVC irradiation as assessed by assays for cell viability and clonogenicity compared to Purα positive control cultures. In reporter plasmid reactivation assays to measure the removal of DNA adducts induced in vitro by UVC, the Purα-negative cells were less efficient in DNA damage repair. Purα-negative cells were also more sensitive to UVC-induced DNA damage measured by Comet assay and showed a decreased ability to remove UVC-induced cyclobutane pyrimidine dimers. In wild-type mouse fibroblasts, expression of Purα is induced following S-phase checkpoint activation by UVC in a similar manner to the NER factor TFIIH. Moreover, co-immunoprecipitation experiments showed that Purα physically associates with TFIIH. Thus, Purα has a role in NER and the repair of UVC-induced DNA damage.Key words: purα, ultraviolet radiation, DNA damage, DNA repair, nucleotide excision repair, TFIIH     

Mutation spectrum in Escherichia coli DNA mismatch repair deficient (mutH) strain

The Dam-directed post-replicative mismatch repair system of Escherichia coli removes base pair mismatches from DNA. The products of the mutH, mutL and mutS genes, among others, are required for efficient mismatch repair. Absence of any of these gene products leads to persistence of mismatches in DNA with a resultant increase in spontaneous mutation rate. To determine the specificity of the mismatch repair system in vivo we have isolated and characterized 47 independent mutations from a mutH strain in the plasmid borne mnt repressor gene. The major class of mutations comprises AT to GC transitions that occur within six base pairs of the only two 5'-GATC-3' sequences in the mnt gene. In the wild type control strain, insertion of the IS1 element was the major spontaneous mutational event. A prediction of the Dam-directed mismatch repair model, that the mutation spectra of dam and mutH strains should be the same, was confirmed.     

5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.     

Overexpression of phospholipase C-gamma1 suppresses UVC-induced apoptosis through inhibition of c-fos accumulation and c-Jun N-terminal kinase activation in PC12 cells.

Ultraviolet-C (UVC) irradiation induces DNA damage and UVC-irradiated cells undergo cell growth arrest to repair the damaged DNA or the induction of apoptosis to prevent the risk of neoplastic transformation. Phospholipase C-gamma1 (PLC-gamma1) is a mediator of growth factor induced-signal cascade, catalyzing the hydrolysis of phosphatidyl 4,5-bisphosphate to generate second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)). PLC-gamma1 is activated by phosphorylation of tyrosine residues upon occupation of cell surface receptors by growth factors and plays an important role in controlling cellular proliferation and differentiation. In this study, we found that PLC-gamma1 was tyrosine phosphorylated within 2.5 min after UVC irradiation. To investigate the role of UVC-induced tyrosine phosphorylation of PLC-gamma1, we compared the effect of UVC between PLC-gamma1 overexpressing cells and empty vector transfected cells. Overexpression of PLC-gamma1 inhibited UVC-induced sub-diploid peak and DNA fragmentation. Northern blot analysis revealed that UVC-induced c-fos mRNA accumulation was inhibited in PLC-gamma1 overexpressing cells, while c-jun expression was not affected. In addition, UVC-induced activation of c-Jun N-terminal kinase (JNK) was significantly suppressed in PLC-gamma1 overexpressing cells. These results suggest that PLC-gamma1 may associate with the protective function against the UVC-induced cell death progression via the inhibition of accumulation of c-fos mRNA and the inhibition of JNK kinase activity.     

Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.     

Studies on mutagenesis and repair induced by platinum analogs

Mutagenesis and cytotoxicity were studied in Escherichia coli by iproplatin and carboplatin, two analogs of cisplatin (CDDP) currently undergoing clinical trial. As with CDDP, mutagenesis by these agents was mediated by the umuDC gene product. In contrast to CDDP, however, mismatch repair did not substantially contribute to survival of cells after exposure to these agents since dam-3 E. coli were not more sensitive than wild type E. coli. UvrA- E. coli, however were more sensitive to these analogs demonstrating that as with CDDP, uvr endonuclease-mediated excision contributes to the repair of DNA damage induced by platinum compounds.     

Involvement of 5-methylcytosine in sunlight-induced mutagenesis.

In human skin cancers, more than 30 % of all mutations in the p53 gene are transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG, found at several mutational hotspots. Since CpGs are methylated along the p53 gene, these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. In Xorder to define the contribution of 5-methylcytosine to sunlight-induced mutations, we have used mouse fibroblasts containing the CpG-methylated lacI transgene as a mutational target. We sequenced 182 UVC (254 nm UV)-induced mutations and 170 mutations induced by a solar UV simulator, along with 75 mutations in untreated cells. Only a few of the mutations in untreated cells were transitions at dipyrimidines, but more than 95% of the UVC and solar irradiation-induced mutations were targeted to dipyrimidine sites, the majority being transitions. After UVC irradiation, 6% of the base substitutions were at dipyrimidines containing 5-methylcytosine and only 2.2% of all mutations were transitions within this sequence context. However, 24% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of them were transitions. Two sunlight-induced mutational hotspots at methylated CpGs correlated with sequences that form the highest levels of cyclobutane pyrimidine dimers after irradiation with sunlight but not with UVC. The data indicate that dipyrimidines that contain 5-methylcytosine are preferential targets for sunlight-induced mutagenesis in cultured mammalian cells, thus explaining the large proportion of p53 mutations at such sites in skin tumors in vivo.     

The PSO3 gene is involved in error-prone intragenic recombinational DNA repair in Saccharomyces cerevisiae

Summary The induction of gene conversion and mitotic crossing-over by photoaddition of psoralens, 254 nm ultraviolet radiation, and nitrogen mustards was determined in diploid cells homozygous for the pso3-1 mutation and in the corresponding wild type of Saccharomyces cerevisiae. For these different agents, the frequency of non-reciprocal events (conversion) is reduced in the pso3-1 mutant compared to the wild type. In contrast, the frequency of reciprocal events (crossing-over) is increased at a range of doses. These observations, together with the block in induced mutagenesis for both reverse and forward mutations previously reported for the pso3-1 mutant, suggest that the PS03 gene product plays a role in mismatch repair of short patch regions. The block in gene conversion in the pso3 homozygous diploid leads, in the case of nitrogen mustards, to specific repair intermediates which are lethal to the cells.     

Mutations induced by ultraviolet light

The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms involving oxidized DNA bases. We also discuss the role of DNA damage-tolerant DNA polymerases in UV lesion bypass and mutagenesis.     

Specificity of N-acetoxy-N-2-acetylaminofluorene-induced frameshift mutation spectrum in mismatch repair deficient Escherichia coli strains mutH, L, S and U

The mismatch repair system of Escherichia coli is known to contribute to the fidelity of the replicational process. This system involves the functions of mutH, mutL, mutS and mutU (uvrD) loci which recognize mispaired bases as a consequence of errors due to the polymerase itself. Chemical modifications of DNA have also been suspected to create mispaired bases which, if the mispaired bases are removed, will lead to mutations by frameshift. Using the pBR322 plasmid DNA modified by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) we have investigated this possibility in a forward mutational assay (tetracycline sensitivity). This fluorene derivative has been shown to induce predominantly frameshift mutations. Our results show that: The sensitivity of the deficient strains mutH, mutL and mutS to the AAF adducts is similar to that of the corresponding wild-type strain. However, the mutU strain appears much more sensitive to those adducts although less than a uvrA, B or C-deficient strain. This suggests that the mutU gene product is involved in the repair of AAF adducts. For the four mut deficient strains, and as it was shown with the wild-type strain, AAF adducts induced mutations to tetracycline sensitivity are only observed when the SOS system of the host bacteria is induced by irradiation of the cells prior to transformation with the modified plasmid. The mutation frequencies depend upon the ultraviolet light doses and similar maxima were found for the four mut strains and the corresponding wild-type strain. In agreement with the results obtained with wild-type or uvrA strains we observe that AAF adducts induce mostly frameshift mutations in the mut strains. Two types of hot spots of mutagenesis were described in wild-type and uvrA strains occurring either at repetitive sequences or at sequences of the type 5' G-G-C-G-C-C 3' (NarI restriction enzyme recognition sequence). While the second type of mutational hot spot does exist in the mismatch repair-deficient strains, we observe that the repetitive sequences are no longer hot spots of mutations in these strains, suggesting that the mismatch repair protein complex is involved in the establishment of AAF-induced frameshift mutations at repetitive sequences.     

Mismatch Repair Deficient Mice Show Susceptibility to Oxidative Stress-Induced Intestinal Carcinogenesis

We have previously established an experimental system for oxidative DNA damage-induced tumorigenesis in the small intestine of mice. To elucidate the roles of mismatch repair genes in the tumor suppression, we performed oxidative DNA damage-induced tumorigenesis experiments using Msh2-deficient mice. Oral administration of 0.2% Potassium Bromate, KBrO₃, effectively induced epithelial tumors in the small intestines of Msh2-deficient mice. We observed a 22.5-fold increase in tumor formation in the small intestines of Msh2-deficient mice compared with the wild type mice. These results indicate that mismatch repair is involved in the suppression of oxidative stress-induced intestinal tumorigenesis in mice. A mutation analysis of the Ctnnb1 gene of the tumors revealed predominant occurrences of G:C to A:T transitions. The TUNEL analysis showed a decreased number of TUNEL-positive cells in the crypts of small intestines from the Msh2-deficient mice compared with the wild type mice after treatment of KBrO₃. These results suggest that the mismatch repair system may simultaneously function in both avoiding mutagenesis and inducing cell death to suppress the tumorigenesis induced by oxidative stress in the small intestine of mice.     

Similarities in sunlight-induced mutational spectra of CpG-methylated transgenes and the p53 gene in skin cancer point to an important role of 5-methylcytosine residues in solar UV mutagenesis

In the p53 gene of human sunlight-associated skin cancers, 35 % of the mutations involve trinucleotide sequences with the rare base 5-methylcytosine (5'PymCG). In order to determine the involvement of 5-methylcytosine in sunlight-induced mutations, we have analyzed the cII transgene in mouse cells, a mutational target gene that we found is methylated at most CpG sequences. We report that the mutational spectra produced by irradiation with 254 nm UVC radiation and simulated sunlight, respectively, differ most dramatically by the much higher involvement of dipyrimidine structures containing 5-methylcytosine in the solar UV mutation spectrum (32 % versus 9 % of all mutations). A distinct mutational hotspot induced by simulated sunlight occurs at a sequence 5'TmCG and is associated with high levels of cis-syn cyclobutane pyrimidine dimer formation. A comparison of sunlight-induced mutational spectra of the cII and lacI transgenes, as well as the p53 gene in skin tumors, shows that 5-methylcytosine is involved in 25 to 40 % of all mutations in all three systems. The combined data make a strong case that cyclobutane pyrimidine dimers forming preferentially at dipyrimidine sequences with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells.