Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes

Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection. The rate of recovery of replicative capacity is inversely related to the initial adduct number. Holding the infected cells temporarily under conditions that prevent viral DNA replication results subsequently in increased recovery, proportional to the holding time. The mechanism of recovery appears to be constitutive and prereplicative. In addition, there is a second mode of recovery which is induced by pretreatment of the host cells with BPDE before infection. The effect of pretreatment is similar to that of extending the holding time before replication: the first molecules begin to replicate earlier but the subsequent rate of recovery is unchanged. The induced mechanism may be either a limited stoichiometric repair process or a slow replicative bypass.     

Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo[a]pyrene-diol-epoxide and dibenzo[a,l]pyrene-diol-epoxide

Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (+/-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the (32)P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.     

Specificity of platinum-DNA adduct repair

Cell lines with resistance to cisplatin and carboplatin often retain sensitivity to platinum complexes with different carrier ligands (e.g., oxaliplatin and JM216). HeLa cell extracts were shown to excise cisplatin, oxaliplatin, and JM216 adducts with equal efficiency, suggesting that nucleotide excision repair does not contribute to the carrier-ligand specificity of platinum resistance. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of the platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts, by the mismatch-repair system that removes newly synthesized DNA opposite Pt-DNA adducts, and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion synthesis. Primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, or JM216 Pt-GG adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma and HIV-1 RT had a similar specificity for translesion synthesis past Pt-DNA adducts (oxaliplatin > or = cisplatin > JM216). In addition, defects in the mismatch-repair proteins hMSH6 and hMLH1 led to increased replicative bypass of cisplatin adducts, but not of oxaliplatin adducts. Finally, primer extension assays performed in the presence of HMG1, which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216). These studies show that DNA polymerases, the mismatch-repair system and damage-recognition proteins can all impart specificity to replicative bypass of Pt-DNA adducts. Replicative bypass, in turn, may influence the carrier-ligand specificity of resistance.     

Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells.

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in the monkey kidney cell line COS7. The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol. Cell. Biol. 4:2227-2230, 1984). When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host. Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid. Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene. The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained. Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions. In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations. Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.     

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.     

An analysis of DNA replication in synchronized CHO cells treated with benzo[a]pyrene diol epoxide

Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.     

XP-A cells complemented with Arg228Gln and Val234Leu polymorphic XPA alleles repair BPDE-induced DNA damage better than cells complemented with the wild type allele

Functional effects of Arg228Gln and Val2343Leu XPA polymorphisms on benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide-(+/-)-anti (BPDE) survival and repair were investigated in SV40 immortalized XP12RO cells complemented with wild type and polymorphic XPA cDNAs in an inducible cDNA expression system. In contrast to previous studies showing little impact of XPA polymorphisms on UV survival and repair, cells complemented with polymorphic XPAs displayed improved BPDE survival and repair as compared to wild type XPA-complemented cells. Survival after BPDE treatment was measured using AlamarBlue reduction and colony forming ability. Cells expressing low levels of either polymorphic XPA had equivalent or improved survival compared to wild type XPA-complemented cells (XPAwt cells). XPA induction improved BPDE survival in Arg228Gln (R228Q cells) and Val234Leu (V234L cells) complemented cells, but not XPAwt cells. BPDE-induced DNA damage repair was measured both by reactivation after transfection of a luciferase reporter plasmid reacted with BPDE in vitro, and by removal of adducts from genomic DNA of BPDE-treated cells. BPDE-induced DNA damage repair in R228Q and V234L cells expressing XPA at very low levels was similar to repair in XPAwt cells expressing XPA at normal levels. XPA induction improved repair in R228Q and V234L cells but not in XPAwt cells. Our findings suggest that both Arg228Gln and Val234Leu XPAs function better than wild type XPA for BPDE adduct removal. These observations differ from UV repair results suggesting that the differences are lesion specific. The location of the polymorphisms within the putative poly(ADP-ribose) binding domain suggests that poly(ADP-ribose) interaction is important in repair.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the beta-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the beta-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.     

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.     

DNA polymerase kappa is specifically required for recovery from the benzo[a]pyrene-dihydrodiol epoxide (BPDE)-induced S-phase checkpoint

Previously we identified an intra-S-phase cell cycle checkpoint elicited by the DNA-damaging carcinogen benzo[a]pyrene-dihydrodiol epoxide (BPDE). Here we have investigated the roles of lesion bypass DNA polymerases polkappa and poleta in the BPDE-induced S-phase checkpoint. BPDE treatment induced the re-localization of an ectopically expressed green fluorescent protein-polkappa fusion protein to nuclear foci containing sites of active DNA synthesis in human lung carcinoma H1299 cells. In contrast, a similarly expressed yellow fluorescent protein-poleta fusion protein showed a constitutive nuclear focal distribution at replication forks (in the same cells) that was unchanged in response to BPDE. BPDE-induced formation of green fluorescent protein-polkappa nuclear foci was temporally coincident with checkpoint-mediated S-phase arrest. Unlike "wild-type" cells, Polk(-/-) mouse embryonic fibroblasts (MEFs) failed to recover from BPDE-induced S-phase arrest, while exhibiting normal recovery from S-phase arrest induced by ionizing radiation and hydroxyurea. XPV fibroblasts lacking poleta showed a normal S-phase checkpoint response to BPDE (but failed to recover from the UV light-induced S-phase checkpoint), in sharp contrast to Polk(-/-) MEFs. The persistent S-phase arrest in BPDE-treated Polk(-/-) cells was associated with increased levels of histone gammaH2AX (a marker of DNA double-strand breaks (DSBs)) and activation of the DSB-responsive kinases ATM and Chk2. These data suggest that in the absence of polkappa, replication forks stall at sites of damage and collapse and generate DSBs. Therefore, we conclude that the trans-lesion synthesis enzyme polkappa is specifically required for normal recovery from the BPDE-induced S-phase checkpoint.     

Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair.

Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide position depends on the interaction between the specific adduct conformation and the local DNA conformation at that nucleotide.     

Activities of human DNA polymerase kappa in response to the major benzo[a]pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion

In cells, the major benzo[a]pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N(2)-dG. In eukaryotes, little is known about lesion bypass of this DNA adduct during replication. Here, we show that purified human Polkappa can effectively bypass a template (+)-trans-anti-BPDE-N(2)-dG adduct in an error-free manner. Kinetic parameters indicate that Polkappa bypass of the (-)-trans-anti-BPDE-N(2)-dG adduct was approximately 41-fold more efficient compared to the (+)-trans-anti-BPDE-N(2)-dG adduct. Furthermore, we have found another activity of human Polkappa in response to the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts: extension synthesis from mispaired primer 3' ends opposite the lesion. In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polbeta and the purified catalytic subunits of yeast Polalpha, Poldelta, and Pol epsilon right before the lesion. Extension by human Polkappa from the primer 3' G opposite the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts was mediated by a -1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polkappa prior to DNA synthesis. Thus, sequence contexts 5' to the lesion strongly affect the fidelity and mechanism of the Polkappa-catalyzed extension synthesis. These results support a dual-function model of human Polkappa in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase.     

Repair of DNA lesions: mechanisms and relative repair efficiencies

DNA is frequently damaged by endogenous agents inside the cells. Some exogenous agents such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and may thus contribute to the 'background' DNA damage in humans. DNA lesions are normally removed by various repair mechanisms. The major repair mechanisms for various DNA lesions are summarized. In contrast to the extensively studied repair mechanisms, much less is known about the relative repair efficiencies of various DNA lesions. Since DNA repair is a crucial defense against carcinogenesis, it may constitute an important factor affecting the carcinogenicity of DNA damaging agents. We have adopted a human cell-free system for measuring relative DNA repair efficiencies based on the concept of repair competition between acetylaminofluorene adducts and other DNA lesions of interest. Using this in vitro system, we determined the relative repair efficiencies of PAH adducts induced by: anti-(+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), anti-(+/-)-benz[a]anthracene-trans-3,4-dihydrodiol-1,2-epoxide (BADE-I), anti-(+/-)-benz[a]anthracene-trans-8,9-dihydrodiol-10, 11-epoxide (BADE-II), anti-(+/-)-benzo[b]fluoranthene-trans-9, 10-dihydrodiol-11,12-epoxide (BFDE), anti-(+/-)-chrysene-trans-1, 2-dihydrodiol-3,4-epoxide (CDE), and anti-(+/-)-dibenzo[a, l]pyrene-trans-11,12-dihydrodiol-13,14-epoxide (DBPDE). While damage by BPDE, DBPDE, CDE, and BFDE were repaired by nucleotide excision repair as efficiently as AAF adducts, the repair of BADE-I and BADE-II adducts were significantly slower in human cell extracts. Damage by DBPDE at 3 microM in vitro yielded approximately 5-fold higher DNA adducts than BPDE as determined by quantitative PCR. This potent DNA reactivity may account in part for the potent carcinogenicity of dibenzo[a,l]pyrene. The correlation of these results to the carcinogenic properties of the PAH compounds is discussed. Furthermore, we show that NER plays a role in AP site repair in vivo in the eukaryotic model organism yeast.     

Ability of adducts formed in a shuttle vector by reactive metabolites of 1-nitropyrene and benzo(a)pyrene to induce mutations when the plasmid replicates in human cells

An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF), was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into human cell line 293, allowed to replicate, and the progeny plasmids rescued and assayed for the frequency of supF mutants. The agents tested were the 7,8-diol-9,10-epoxide of benzo(a)pyrene (BPDE) and 1-nitrosopyrene (1-NOP). With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies 15 to 25 times higher than the background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately four times more mutagenic than 1-NOP, which binds principally at the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule, but it could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least three times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base substitutions. DNA sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmid and 60 from 1-NOP-treated plasmid indicated that 70% to 80% contained a single base substitution, 5%-10% had two base substitutions, and 4%-10% had small insertions or deletions (one or two basepairs). The majority (83%) of the base substitutions in mutants from BPDE- or 1-NOP-treated plasmid were transversions, mainly G.C----T.A. Each carcinogen produced its own spectrum of mutations.     

Repair of benzo[a]pyrene diol epoxide damaged bacteriophage T7 DNA determined by survival of phage made by in vitro packaging

DNA from bacteriophage T7 was treated with benzo[a]pyrene diol epoxide (BPDE) and the number of covalently bound adducts per T7 genome was determined. BPDE treated T7 DNA was then incubated in an in vitro DNA packaging system so as to form infective T7 phage. The observed reduced survival of these phage measured with Escherichia coli uvrA- indicator bacteria showed that the BPDE treated DNA was in fact utilized by the in vitro packaging system and that the resulting phage contained DNA damage caused by in vitro exposure to BPDE. T7 DNA damage by BPDE was also incubated in an in vitro DNA repair system that used partially purified uvrABC proteins from E. coli. Alkaline sucrose gradient analysis demonstrated that nicks were introduced into the damaged DNA and that these incisions were repaired to yield nearly intact DNA molecules of about the size of a T7 genome. Encapsulation of the repaired DNA with the packaging system yielded phage that showed higher survival than the unrepaired control when plated on uvrA- indicator bacteria.