Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct. On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v. mutagenesis.     

(6-4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers

A polyclonal antiserum raised against UV-irradiated DNA can be used to assay cyclobutane pyrimidine dimers and Pyr(6-4)Pyo photoproducts specifically by changing the nature of the 32P-labelled antigen. Pyr(6-4)Pyo photoproducts were removed faster than cyclobutane dimers in UV-irradiated human, hamster and mouse cells. Xeroderma pigmentosum cells from complementation groups A, C and D were deficient in the repair of both lesions.     

DNA sequence context affects UV-induced mutagenesis in Escherichia coli

We investigated the effect of altering the DNA sequence surrounding a mutable target site on the production of ultraviolet light (UV) induced mutations. Site-directed base substitutions were incorporated on both sides of a TAA sequence encoding a UAA nonsense defect in the tyrA14 allele of Escherichia coli. This allele is readily revertable by UV and a total of eight different base substitution mutations can be recovered. Five different strains harboring DNA sequences allowing the formation of 5'-TT, 5'-CT and 5'-TA* photoproducts were constructed and exposed to UV. DNA sequence analysis was used to determine the spectrum of the revertants that were recovered. The results showed that changes at the 3'-base of a TT site were predominantly T to C transitions and T to A transversions. However, unlike the TT site, a 5'-CT site produced a relatively high frequency of T to G transversions. In addition, T to A transversions that could not have been targeted by a cyclobutane-type or [6-4]-type pyrimidine dimer were produced; this result suggested that these mutations may be targeted by a TA* photoproduct. Also, a distinct strand bias was noted for two mechanistically identical base substitutions in a strain having a palindromic target sequence; this result may reflect an unequal damage distribution or processing of photoproducts as a consequence of asymmetric DNA replication. Finally, our results show that DNA sequences expected to allow the greatest density of UV-induced DNA damage produce the highest mutation frequencies. Overall, these findings provide new insights regarding the role of DNA photoproducts in UV mutagenesis.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

Base substitution mutations induced in the cI gene of lambda phage by neocarzinostatin chromophore: correlation with depyrimidination hotspots at the sequence AGC.

Treatment of intact lambda phage with the nonprotein chromophore of neocarzinostatin resulted in efficient phage inactivation and generation of clear-plaque mutants. Both effects required a preincubation at low pH to allow diffusion of chromophore into the phage head. Chromophore activation was then effected by addition of a sulfhydryl cofactor, followed by a shift to neutral pH. Sequence analysis of mutations mapped to the DNA-binding region of the cI gene revealed that nearly all were single base substitutions. Significant numbers of all possible base changes were found, with A:T to G:C transitions being the most frequent events. Of 11 G:C to A:T transitions, 7 were found at C residues in the trinucleotide sequence AGC, which has previously been shown to be a hotspot for chromophore-induced depyrimidination. This result, as well as the SOS dependence of mutagenesis and the overall distribution of various types of base substitutions, is consistent with the hypothesis that apurinic/apyrimidinic sites are important mutagenic lesions.     

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

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

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3''----5'' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).     

UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand.

The molecular nature of 254 nm ultraviolet light (UV)-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in UV24 Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, was determined. Sequence analysis of 19 hprt mutants showed that single base substitutions (9 mutants) and tandem base changes (7 mutants) dominated the UV mutation spectrum in this cell line. Sixty-five percent of the base substitutions were GC greater than AT transitions, whereas the rest consisted of transitions and transversions at AT base pairs. Analysis of the distribution of dipyrimidine sites over the two DNA strands, where the photoproducts causing these mutations presumably were formed, showed that 12 out of 14 mutations were located in the transcribed strand of the hprt gene. A similar strand distribution of mutagenic photoproducts as in UV24 has previously been found in two other UV-sensitive Chinese hamster cell lines (V-H1 and UV5), indicating that under defective nucleotide excision repair conditions the induction of mutations is strongly biased towards lesions in the transcribed strand of the hprt gene. A plausible explanation for this phenomenon is that during DNA replication large differences exist in the error rate with which DNA polymerase(s) bypass lesions in the templates for the leading and lagging strand, respectively.     

Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.     

Mutagenic specificity of ultraviolet light

Genetic and sequencing studies of ultraviolet light (u.v.)-induced mutations in the lacI gene of Escherichia coli show the following: u.v. stimulates many types of mutations. In lacI, base substitutions account for 60 to 65% of the observed mutations, small frameshifts 30 to 35%, and deletions of more than several base-pairs approximately 5%. A comparison of the mutational spectrum of u.v.-induced mutations with those of other SOS-dependent mutagens and with the mutations produced by inducing the SOS system in the absence of mutagenic treatment indicates that most u.v.-induced base substitutions are "targeted", resulting from premutational lesions across from the site of the mutations. Among base substitutions, both transitions and transversions occur, although the most favored mutational sites involve G X C----A X T transitions. G X C----A X T transitions are induced preferentially at sites of adjacent pyrimidines. In one case the conversion of a site from -A-C-A- to -T-C-A- results in a 15-fold increase in u.v.-induced C----T transitions. Frameshifts at certain sites are well-induced by u.v., and the largest hotspot in the I gene involves the loss of an (sequence in text) base pair from a (sequence in text) sequence. Of 25 frameshifts detected by DNA sequencing, 23 mutations at seven different sites result from the elimination of a single base-pair, and two mutations result from the elimination of two base-pairs. No additions were detected. The use of a lacI-Z fusion system, which allows direct selection of frameshifts of either sign, reveals that throughout the entire gene frameshifts that eliminate a single base-pair (-1) predominate by a factor of 20 or more over frameshifts that add a single base-pair (+1). In one case a two-base-pair elimination occurs frequently, resulting in the loss of a -C-T- sequence (on one strand), or a -T-C- sequence, from a -C-T-C-T-C-T-C- sequence. For both frameshifts and base substitutions, some aspect of the larger surrounding sequence beyond the nearest neighbors can influence mutation rates by as much as 50-fold, thus determining which sites are seen as hotspots. The bearing of these and other data on the detailed mechanism of mutagenesis is considered in the Discussion.     

Effect of photoreactivation on mutagenesis of lambda phage by ultraviolet light

There is disagreement in the literature as to whether the major mutagenic photoproduct induced in DNA by ultraviolet light is the cyclobutane dipyrimidine dimer, the most common product, or the [6-4] photoproduct, the next most frequent. In the experiments reported here, cyclobutane dimers were removed from irradiated lambda phage DNA by enzymatic photoreactivation, a process thought to affect no other photoproduct. Photoreactivation of lambda phage in host cells and of lambda DNA in solution reduced clear plaque mutants per plaque-forming unit by two-thirds, in host cells with a constant and near-maximal expression of the SOS functions required for mutagenesis. This result is interpreted to mean that removal of cyclobutane dimers in or near the mutated gene reduces mutation induced by ultraviolet light by two-thirds; therefore, cyclobutane dimers in the phage DNA are responsible for most observed mutations. DNA sequences of mutations in photoreactivated phage showed a smaller fraction of G.C to A.T transitions and a larger fraction of A.T to G.C transitions, compared to phage that were not photoreactivated. This suggests that cyclobutane dimers at TC and CC sites are particularly mutagenic.     

The DNA of UV-irradiated normal and excision-deficient mammalian cells undergoes relaxation in an initial stage of DNA repair

Using a radioimmunoassay specific for Pyr(6-4)Pyo photoproducts, we have demonstrated the removal of these lesions from denaturated DNA isolated from UV-irradiated Chinese hamster ovary cells at various times post irradiation. When assayed undenatured, these same DNA samples, which are initially 10-20 times less capable of binding antibody, show a substantial increase in binding capacity during the first few hours of repair. At 3 h post irradiation the difference between native and heat-denatured DNA samples is negligible, indicating that all of the residual lesions are contained in a single-stranded (relaxed) configuration. This relaxation also occurs in UV-hypersensitive cell lines, that are deficient in the ability to remove Pyr(6-4)Pyo photoproducts. Novobiocin, an inhibitor of topoisomerase II, prevents both the initial increase in binding and the subsequent excision of the antibody-binding sites.     

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.     

Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome. Sequences of mutants in the cI gene of a lambda lysogen

DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E. coli chromosome. The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage. The cause of this difference is not known. Of 17 frameshifts in phage and prophage, six have an accompanying base substitution. These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism.     

Excision repair influences the site and strand specificity of sunlight mutagenesis in yeast.

A collection of 384 mutations recovered in a tRNA gene (SUP4-o) following exposure of isogenic excision-repair-proficient (RAD1) or deficient (rad1) strains of the yeast Saccharomyces cerevisiae to sunlight was characterized by DNA sequencing. In each case, greater than 90% of the mutations were single base-pair substitutions with events at G.C pairs constituting most of the changes. However, more than half of these substitutions were transversions in the RAD1 strain whereas transitions predominated in the rad1 strain. Tandem double substitutions were recovered in both strains and the individual changes were exclusively G.C----A.T transitions. The majority of single substitutions, and all tandem double changes, were at base-pairs where the pyrimidine(s) was part of a dipyrimidine sequence and the site specificities were consistent with cyclobutane dimers and/or pyrimidine (6-4) pyrimidone photoproducts contributing to sunlight mutagenesis. Yet, the data also pointed to an important role for lesions that form at G.C pairs and give rise to transversions. Analysis of the strand specificity of sunlight mutagenesis indicated that transitions or transversions at G.C pairs occurred preferentially in SUP4-o at sites where a dipyrimidine or a guanine, respectively, was on the transcribed strand. These biases required a functional excision-repair system.     

The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions.

4-Nitroquinoline-1-oxide is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. In ds or ss damaged DNA the ratio C8/N2 adducts is 1:2 and 8-10:1, respectively. In bacteria and yeast 4NQO has been shown to be a base substitution mutagen acting at G residues inducing mainly G to A transitions. We determined the mutational spectrum induced by the 4NQO metabolite, acetoxy-4-aminoquinoline 1-oxide, in the M13lacZ'/E. coli lacZ delta M15 alpha complementation assay using ssDNA. Among 68 Ac-4HAQO induced mutants, G to Pyr transversion was the most frequent base substitution observed. By comparison with dsDNA based systems, our data suggest that dGuo-C8-AQO induces G to Pyr transversions. A mechanism to explain how this lesion may induce transversions is proposed.     

Mutagenic properties of the 8-amino-2'-deoxyguanosine DNA adduct in mammalian cells.

The DNA adduct 8-amino-2'-deoxyguanosine (8-amino-dG) is found in liver DNA of rats treated with the hepatocarcinogen 2-nitropropane. Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of 8-amino-dG in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'-TCCTCCTX1G2CCTCTC and 5'-TCCTCCTG1X2CCTCTC, X = dG or 8-amino-dG) with the lesion positioned at codon 60 or 61 of the non-coding strand of the human c-Ha- ras1 gene were inserted into single-stranded phagemid vectors and transfected into COS-7 cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequencing to establish the mutation frequency and spectrum produced by the modified base. The correct base, dCMP, was incorporated preferentially opposite 8-amino-dG at X1and X2. When 8-amino-dG was at X1, targeted GNH2-->T transversions were detected, along with smaller numbers of GNH2-->A transitions and GNH2-->C transversions. When the adduct was at X2, only GNH2-->T transversions were observed. The frequencies of targeted mutation at X1and X2were 2.7 and 1.7%, respectively. Mutation frequency and mutagenic spectrum were sequence context dependent. In addition, non-targeted G-->T transversions, accompanied by some G-->A transitions, were detected 5' to 8-amino-dG when the lesion was at X2. We conclude that 8-amino-dG is a mutagenic lesion, generating G-->T and G-->C transversions and G-->A transitions in mammalian cells.     

Abnormal, Error-Prone Bypass of Photoproducts by Xeroderma Pigmentosum Variant Cell Extracts Results in Extreme Strand Bias for the Kinds of Mutations Induced by UV Light

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C→T, 30% of the base substitutions consist of C→A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C→T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZα, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to ~37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but ~2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C→A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T→A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.     

Structural determination of the ultraviolet light-induced thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct.

Ultraviolet light induces damage to DNA, with the majority of the damage expressed as the formation of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The (6-4) photoproducts have been implicated as important UV light-induced premutagenic DNA lesions. The most abundant of the (6-4) products is the thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct, or TC (6-4) product. The structure of the TC (6-4) product was deduced by proton NMR, IR, and fast atom bombardment mass spectroscopy, and the product was found to differ from the previously described photoadduct, Thy(6-4)Pyo, by the presence of an amino group at the 5 position of the 5' pyrimidine. The implications of this structure on DNA base pairing and the induction of ultraviolet light-induced mutations are discussed.     

Mechanisms of Mutagenesis by Chloroacetaldehyde

A number of bifunctional chemical mutagens induce exocyclic DNA lesions. For example, 2-chloroacetaldehyde (CAA), a metabolite of vinyl chloride, readily reacts with single-stranded DNA to predominantly form etheno lesions. Here, we report on in vivo mutagenesis caused by CAA treatment of DNA in vitro. These experiments used partially duplex phage M13AB28 replicative form DNA in which a part of the lacZ gene sequence was held in single-stranded form to direct reaction with CAA. CAA-treated partial duplex DNA was transfected into Escherichia coli, and the induced base changes were defined by DNA sequencing. These experiments suggested that CAA treatment induced mutations at cytosines, much less efficiently at adenines, but not at guanines or thymines. Among mutations targeted to cytosine, 80% were C-to-T transitions and 20% were C-to-A transversions. Application of a post-labeling method detected dose-dependent formation of ethenoadenine and ethenocytosine in CAA treated DNA. These data indicate that ethenocytosine is a highly efficient mutagen with properties suggestive of a non-instructional DNA lesion in vivo. Paradoxically, ethenoadenines are efficiently bypassed by a mechanism which appears to be largely nonmutagenic.