Relationship between DNA Methylation and Mutational Patterns Induced by a Sequence Selective Minor Groove Methylating Agent
1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês
10.1074/jbc.274.26.18327
ISSN1083-351X
AutoresJack Kelly, Alberto Inga, Fa-Xian Chen, Prasad Dande, Dharini Shah, Paola Monti, Anna Aprile, Philip A. Burns, Gina B. Scott, Angelo Abbondandolo, Barry Gold, Gilberto Fronza,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoMe-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generateN 3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT → TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600–602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10−6, Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner. Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generateN 3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT → TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600–602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10−6, Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner. Most carcinogens and alkylating antineoplastic agents react with DNA to afford a diverse mixture of DNA lesions (1, 2Beranek D.T. Weis C.C. Swenson D.H. Carcinogenesis. 1980; 1: 595-605Crossref PubMed Scopus (342) Google Scholar). This complexity is a barrier to being able to quantitatively and qualitatively dissect out the biological role(s) of the individual DNA lesions relative to the mutagenic and/or toxic potency of the agent. Most methylating agents, for example, transfer methyl groups to as many as 10 nucleophilic nitrogens and oxygens in DNA (2Beranek D.T. Weis C.C. Swenson D.H. Carcinogenesis. 1980; 1: 595-605Crossref PubMed Scopus (342) Google Scholar).O 6-Methylguanine represents one of the few lesions extensively studied for which the mutagenic and toxic potential have been documented in detail (3Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar). In order to exercise some regulation over the alkylation pattern, we have prepared groove and sequence selective DNA damaging agents that provide significant control over the types of DNA lesions that are generated (4Konakahara T. Wurdeman R.L. Gold B. Biochemistry. 1988; 27: 8606-8613Crossref PubMed Scopus (22) Google Scholar, 5Church K.M. Wurdeman R.L. Zhang Y. Chen F.-X. Gold B. Biochemistry. 1990; 29: 6827-6838Crossref PubMed Scopus (33) Google Scholar, 6Zhang Y. Chen F.-X. Mehta P. Gold B. Biochemistry. 1993; 32: 7954-7965Crossref PubMed Scopus (64) Google Scholar, 7Mehta P. Church K. Williams J. Chen F.-X. Encell L. Shuker D.E.G. Gold B. Chem. Res. Toxicol. 1996; 9: 939-948Crossref PubMed Scopus (12) Google Scholar). One such compound is Me-lex 1The abbreviations used are: Me-lex, {1-methyl-4-[1-methyl4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2-carboxamido}propane; 3-A, N3 position of adenine; MMS, methyl methanesulfonate ester; MNU, N-methyl-N-nitrosourea; CENU(s), N-chloroethyl-N-nitrosourea(s); lex, lexitropsin; N 3-Alkyl-A, N 3-alkyladenine; CCNU, N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea; O 6-Alkyl-G, O 6-alkylguanine; 3-MeA, N 3-methyladenine; 3-MeG, N 3-methylguanine; 7-MeG, N 7-methylguanine; EC, electrochemical; HPLC, high performance liquid chromatography; Me, methyl; PCR, polymerase chain reaction.(Fig.1), which is a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide (6Zhang Y. Chen F.-X. Mehta P. Gold B. Biochemistry. 1993; 32: 7954-7965Crossref PubMed Scopus (64) Google Scholar). The dipeptide equilibrium binds in the minor groove of DNA at (A/T)n sequences (5Church K.M. Wurdeman R.L. Zhang Y. Chen F.-X. Gold B. Biochemistry. 1990; 29: 6827-6838Crossref PubMed Scopus (33) Google Scholar, 6Zhang Y. Chen F.-X. Mehta P. Gold B. Biochemistry. 1993; 32: 7954-7965Crossref PubMed Scopus (64) Google Scholar). Based on empirical observation, Me-lex has the following binding preference: 5′-ATT > TTA > TAA (6Zhang Y. Chen F.-X. Mehta P. Gold B. Biochemistry. 1993; 32: 7954-7965Crossref PubMed Scopus (64) Google Scholar). Reactivity at those sites is consistent with the mode of lex-DNA interaction determined by a series of structural studies (cited in Ref.6Zhang Y. Chen F.-X. Mehta P. Gold B. Biochemistry. 1993; 32: 7954-7965Crossref PubMed Scopus (64) Google Scholar). While such information on lex binding to DNA provides a basis to rationalize experimentally verified binding domains, it is not possible to predict a priori which A/T-rich regions will be preferred binding sites within large DNA fragments. As a consequence of the dipeptide's binding affinity, the Me-lex efficiently methylates DNA in the minor groove at the N3 position of adenine (3-A) (6Zhang Y. Chen F.-X. Mehta P. Gold B. Biochemistry. 1993; 32: 7954-7965Crossref PubMed Scopus (64) Google Scholar,8Encell L. Shuker D.E.G. Foiles P.G. Gold B. Chem. Res. Toxicol. 1996; 9: 563-567Crossref PubMed Scopus (38) Google Scholar). This contrasts with the methylation pattern induced by simple methylating agents such as methyl methanesulfonate ester (MMS), dimethyl sulfate, and N-methyl-N-nitrosourea (MNU), which predominantly yield the major grooveN 7-methylguanine (7-MeG) adduct (2Beranek D.T. Weis C.C. Swenson D.H. Carcinogenesis. 1980; 1: 595-605Crossref PubMed Scopus (342) Google Scholar). For comparison, the ratio of 3-MeA to 7-MeG is approximately 1:10 for MMS based on in vivo and in vitro assays (1, 2Beranek D.T. Weis C.C. Swenson D.H. Carcinogenesis. 1980; 1: 595-605Crossref PubMed Scopus (342) Google Scholar), while it changes to 100:1 for Me-lex (8Encell L. Shuker D.E.G. Foiles P.G. Gold B. Chem. Res. Toxicol. 1996; 9: 563-567Crossref PubMed Scopus (38) Google Scholar). The strong methylation preference of Me-lex for 3-A is also observed when it is incubated with cells in culture: under these conditions the only lesion detected is 3-MeA while MMS gives the traditional 3-MeA to 7-MeG 1:10 ratio (9Engelward B.P. Allan J.M. Dreslin A.J. Kelly J.D. Wu M.M. Gold B. Samson L.D. J. Biol. Chem. 1998; 273: 5412-5418Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The significant increase in 3-MeA formation closely parallels the increased cytotoxicity of Me-lex versus MMS. This is consistent with the observation that Me-lex cytotoxicity is magnified in alkyladenine-DNA glycosylase (Aag) null ES cells (10Engelward B.P. Dreslin A. Christensen J. Huszar D. Kurahara C. Samson L.D. EMBO J. 1996; 15: 945-952Crossref PubMed Scopus (184) Google Scholar) and in repair-deficient (tagA) bacteria (11Dinglay S. Gold B. Sedgwick B. Mutat. Res. 1998; 407: 109-116Crossref PubMed Scopus (34) Google Scholar). The Aag protein repairs a variety of DNA lesions including 3-alkyladenine (12Roy R. Brooks C. Mitra S. Biochemistry. 1994; 33: 15131-15140Crossref PubMed Scopus (51) Google Scholar) while tagA is specific for 3-alkyladenine. Recently, by combining a chemical and a genetic approach it has been shown that unrepaired 3-MeA have the potential to induce sister chromatid exchanges, chromosome aberrations, cell cycle arrest at the G1/S boundary, p53 induction, and apoptosis (9Engelward B.P. Allan J.M. Dreslin A.J. Kelly J.D. Wu M.M. Gold B. Samson L.D. J. Biol. Chem. 1998; 273: 5412-5418Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), but exactly how this is achieved is not yet clear. Those combined results point toward 3-MeA being mainly a lethal lesion (10Engelward B.P. Dreslin A. Christensen J. Huszar D. Kurahara C. Samson L.D. EMBO J. 1996; 15: 945-952Crossref PubMed Scopus (184) Google Scholar,13Karran P. Lindahl T. Ofsteng I. Evensen G. Seeberg E. J. Mol. Biol. 1980; 140: 101-127Crossref PubMed Scopus (104) Google Scholar). If this were true the 3-MeA specific inducer Me-lex can be considered a new potential antineoplastic agent. Furthermore, it might be expected that such a compound would combine a high cytotoxicity and a low mutagenicity, avoiding (or diminishing) the undesired and well documented carcinogenic property of alkylating agents used in cancer therapy (14Henry-Amar M. Dietrich P.Y. Hematol. Oncol. Clin. N. Am. 1993; 7: 369-387Abstract Full Text PDF PubMed Google Scholar, 15Marselos M. Vainio H. Carcinogenesis. 1991; 12: 1751-1766Crossref PubMed Scopus (78) Google Scholar). Therefore, Me-lex may potentially have a higher therapeutic index with respect to other antineoplastic compounds. In this light, we wanted to investigate the relationship between A-specific DNA lesions and mutations induced by Me-lex at the nucleotide level. Using the human p53 tumor suppressor gene cDNA inserted in a yeast expression vector as a target (16Inga A. Iannone R. Monti P. Molina F. Bolognesi M. Abbondandolo A. Iggo R. Fronza G. Oncogene. 1997; 14: 1307-1313Crossref PubMed Scopus (39) Google Scholar), the sequence specificity for DNA alkylation by Me-lex was determined through the conversion of the adducted sites into single strand breaks by sequential neutral thermal hydrolysis and exposure to base. These results are compared with the lethal and mutagenic effects of Me-lex using an in vitro mutagenesis protocol for plasmid DNA modification, and exploiting a DNA repair-proficient haploidSaccharomyces cerevisiae strain for the processing of DNA lesions into mutations (17Flaman J.M. Frebourg T. Moreau V. Charbonnier F. Martin C. Chappuis P. Sappino A.-P. Limacher J.-M. Bron L. Benhattar J. Tada M. van Meir E.G. Estreicher A. Iggo R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar). Unless stated otherwise, reagents of the highest purity were purchased from Sigma or Aldrich (Milwaukee, WI). Me-lex was prepared as described previously (6Zhang Y. Chen F.-X. Mehta P. Gold B. Biochemistry. 1993; 32: 7954-7965Crossref PubMed Scopus (64) Google Scholar). Restriction enzymes, phosphatases, kinases, and DNA polymerases were obtained from New England Biolabs (Beverly, MA). Calf thymus DNA (1 mm) was incubated with 100 μm Me-lex or 5 mm MMS in 10 mm buffer Tris-HCl buffer (pH 7.6) for 24 h at room temperature, in the presence or absence of 100 μm distamycin. The DNA was precipitated, washed, and dissolved in Tris-HCl buffer (pH 7.0). The solution was heated at 100 °C for 30 min to selectively release N-alkylpurines from DNA. The solution was then treated with ice-cold 0.1 nHCl to precipitate the DNA, and the supernatant collected and analyzed for 3-MeG, 7-MeG, and 3-MeA using reverse phase HPLC: column, ODS C18 YMC (5 mm × 25 cm); column temperature, 40 °C; solvent: 95% 0.1 mNaOAc (pH 5.2), 5% MeOH; UV (λ, 270 nm) and electrochemical (EC) (3-MeG and 7-MeG) detection. The preparation of the 5′-32P-labeled restriction fragments from the plasmid DNA was performed as described previously (18Inga A. Chen F.-X. Monti P. Aprile A. Campomenosi P. Menichini P. Ottaggio L. Viaggi S. Abbondandolo A. Gold B. Fronza G. Cancer Res. 1999; 59: 689-695PubMed Google Scholar). Briefly, plasmid pLS76 containing the p53 cDNA, was restricted to obtain fragments including positions 169 to 1037 (PshAI site (position 169), NcoI site (position 476), Bsu36I (position 667), StuI (position 1037)). The PshAI/NcoI DNA fragment (5′-32P-labeled on the transcribed (noncoding) strand at the NcoI site) was obtained using NcoI restriction endonuclease, phosphatase, and kinase treatments, followed by a final PshAI digestion and polyacrylamide gel purification. The 5′-32P-end-labeledNcoI/Bsu36I DNA fragment (5′-32P-labeled on the nontranscribed (coding) strand at the NcoI site) was obtained by initial endonuclease restriction with NcoI, followed by sequential treatment with calf intestine alkaline phosphatase, phosphorylation with T4 kinase in the presence of [γ-32P]ATP. The DNA was then restricted with Bsu36I and the labeled fragment isolated from a 6% polyacrylamide gel. The same procedure was used to prepare the 5′-32P-end-labeled Bsu36I/NcoI DNA fragment (5′-32P-labeled on the transcribed (noncoding) strand at the Bsu36I site), except that the order of enzyme digestion was reversed. The 5′-32P-end-labeledStuI/Bsu36I DNA fragment (5′-32P-labeled on the transcribed (noncoding) strand at the StuI site) was obtained by initial endonuclease restriction with StuI, followed by sequential treatment with calf intestine alkaline phosphatase, phosphorylation with T4 kinase in the presence of [γ-32P]ATP. The DNA was then restricted with Bsu36I and the labeled fragment isolated from a 6% polyacrylamide gel. The p53 5′-labeled restriction fragment (200,000 cpm) and sonicated calf thymus DNA (83 μm final concentration) in 10 mm Tris, 1 mm EDTA buffer (pH 7.6) were incubated with Me-lex (final concentrations in figure legends) in the presence or absence of 100 μm distamycin for 2 h at 37 °C. The DNA was EtOH/NaOAc precipitated, and washed with cold 70% EtOH. The DNA was dissolved in 30 μl of 10 mm Tris buffer (pH 7.0), and heated at 90 °C for 15 min. The DNA was then re-precipitated, washed as described above, and dried under vacuum. The pellet was resuspended in 100 μl of 1 mpiperidine and heated at 90 °C for 30 min. The piperidine was removed by repeated lyophilization and the DNA resuspended in 10 μl of 80% formamide (v/v), 50 mm Tris borate buffer (pH 8.3), 1 mmEDTA, 0.1% xylene cyanol (w/v), and 0.1% bromphenol blue (w/v). The radioactivity of an aliquot from each sample was determined; the remaining sample was denatured (3 min at 90 °C) and then chilled in ice-water. An equivalent amount of radioactivity for each reaction was loaded on a 12% polyacrylamide denaturing gel (7.78 murea) and run at 65 W constant power. For sequence markers, lanes containing Maxam-Gilbert G and G + A (and in some cases C and C + T) reactions were included in each gel. The gels were dried and analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). In all cases cleavage bands were assigned by underexposing the gel so that the identification of the site was unequivocal. The yeast expression vector pLS76 harboring a human wild-type p53 cDNA under the control of an ADH1 promoter and containing the LEU2 selectable marker, the plasmid pRDI-22 used for gap repair assay, and the haploid S. cerevisiae strain yIG397 (MATa ade2-1 leu2-3, 112 trp1-1 his3-11, 15 can1-100 ura3-1 URA3 3xRGC::pCYC1::ADE2) used as recipient of pLS76 and for gap repair assays were previously described (16Inga A. Iannone R. Monti P. Molina F. Bolognesi M. Abbondandolo A. Iggo R. Fronza G. Oncogene. 1997; 14: 1307-1313Crossref PubMed Scopus (39) Google Scholar). The p53-dependent reporter ADE2 gene allowed the phenotypic selection of p53 mutants (17Flaman J.M. Frebourg T. Moreau V. Charbonnier F. Martin C. Chappuis P. Sappino A.-P. Limacher J.-M. Bron L. Benhattar J. Tada M. van Meir E.G. Estreicher A. Iggo R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar). Standard yeast manipulations were performed as described (19Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar). Me-lex was dissolved in absolute EtOH immediately before the treatment. 1.5 μg of plasmid pLS76 DNA was treated with different Me-lex concentrations in 10 mm Tris HCl (pH 7.4), 1 mm EDTA, 50% EtOH for 1 h at 37 °C. DNA was purified by 3 EtOH precipitations, washed with 70% EtOH, and resuspended in sterile water. Damaged or undamaged vectors were then transfected into yIG397 cells by electroporation and transformants were plated on selective synthetic medium containing 1 m sorbitol. After 3 days of incubation at 30 °C colonies appeared. The selection for the plasmid marker (LEU2) allowed an indirect determination of the lethal effect of the damaging treatment as the number of transformants scored in transfections with damaged plasmids with respect to that obtained with undamaged vector. As transformation plates contained a minimal amount of adenine, adenine auxotrophs were able to achieve only a few cell divisions and thus produced smaller red colonies. Spontaneous and induced mutant frequency were defined as the number of small red colonies with respect to the total number of transformants. The fold of mutant induction was defined as the ratio between the mutant frequency observed with damaged vector with respect to the spontaneous frequency. Phenotypic mutant clones were purified and characterized at the molecular level as described previously (16Inga A. Iannone R. Monti P. Molina F. Bolognesi M. Abbondandolo A. Iggo R. Fronza G. Oncogene. 1997; 14: 1307-1313Crossref PubMed Scopus (39) Google Scholar). Only independent mutants were included in the mutation spectrum; those carrying the same genetic alterations were considered independent only if they were isolated from different transfections. To study the biological consequences of 3-MeA, a synthetic compound was developed to target alkylation to the N3 position of adenine (3-A). Me-lex is composed of an S N2 methyl donating domain and a lexitropsin dipeptide, which equilibrium binds in the minor groove at A:T-rich regions in double-stranded DNA. Specific minor groove binding at A:T base pairs limits alkylation to the minor groove atoms and enhances alkylation at 3-A that are in a lex-binding domain. After in vitro DNA alkylation with Me-lex (100 μm) and MMS (5 mm), the yields of the major (7-MeG) and the minor groove (3-MeG and 3-MeA) adducts were determined using reverse phase HPLC coupled with UV and EC detection. EC detection was required to measure the 3-MeG and 7-MeG adducts when formed in very low yields. More than 99% of the lesions induced by Me-lex were 3-MeA, while 7-MeG represents the major adduct induced by MMS (82%) (TableI). Similar results were obtained with MNU (data not shown). The absolute amount of 3-MeG was the same for the two methylating agents. The specificity for alkylation at adenines by Me-lex was judged by determining the relative abundance of 3-MeAversus 7-MeG. As expected, the ratio of 3-MeA to 7-MeG was >120:1 for Me-lex while it was 1:5 for MMS. Thus, the relative abundance of minor to major groove adduction is ∼600-fold increased with Me-lex compared with MMS. Assuming a linear dose/lesion relationship, it can be calculated that at equimolar concentration Me-lex caused a 1500-fold increase in the absolute yield of 3-MeA and approximately a 30-fold increase in minor groove G-lesion compared with MMS. Therefore, it appears that while Me-lex may enhance the delivery of methyl groups to potential nucleophilic atoms in the minor groove, there is an overwhelming selection for the 3-A site. Interestingly, the co-addition of distamycin, which binds to the same recognition sites as the lex peptide (5Church K.M. Wurdeman R.L. Zhang Y. Chen F.-X. Gold B. Biochemistry. 1990; 29: 6827-6838Crossref PubMed Scopus (33) Google Scholar), quantitatively inhibited methylation by both Me-lex and MMS at all minor groove sites, i.e. 3-MeA and 3-MeG (Table I).Table IIn vitro methylation of calf thymus DNACompoundAdduct yieldsaPicomoles of adduct/μg of DNA.Adduct ratio3-mA7-mG3-mG3-mA/7-mG3-mA/3-mGmmMeOSO2(CH2)2-lexbIncubation time, 24 h at ambient temperature.0.120.35 ± 1.710.16 ± 0.030.05 ± 0.01127.2407.00.1 + 0.1 DistamycinNDcND, not detected, i.e. less than, 0.008 and 0.005 pmol of adduct/μg of DNA for 3-mA and 3-mG, respectively.0.24 ± 0.16NDMMSbIncubation time, 24 h at ambient temperature.5.00.68 ± 0.083.47 ± 0.340.08 ± 0.020.28.55.0 + 0.1 DistamycinND3.54 ± 0.24NDa Picomoles of adduct/μg of DNA.b Incubation time, 24 h at ambient temperature.c ND, not detected, i.e. less than, 0.008 and 0.005 pmol of adduct/μg of DNA for 3-mA and 3-mG, respectively. Open table in a new tab The sequence-dependent DNA alkylation by Me-lex in different restriction fragments is shown in Figs.Figure 2, Figure 3, Figure 4, Figure 5. The assignment of the cleavage sites was done using gels analyzed under conditions of lower exposure. In order to generate strand breaks at potential 7-MeG and 3-MeA adduction sites, the DNA was subjected to neutral thermal hydrolysis. This procedure to release N-alkylpurine lesions is identical to that described for the quantitative HPLC adduct analysis. Consistent with the HPLC-based adduct studies, the sequencing gels demonstrate that DNA methylation by Me-lex was almost exclusively at A, with virtually no G bands being observed.Figure 4Cleavage of the 5′-32P-end-labeledBsu36I/NcoI DNA fragment (5′-32P-labeled (*) on the nontranscribed (coding) strand at the NcoI site) of the p53 cDNA, induced by Me-lex at the indicated concentrations in the presence or in the absence of distamycin (dis), by sequential heating at neutral pH and exposure to hot piperidine. Control,undamaged DNA subjected to sequential heating at neutral pH and exposure to hot piperidine; G and G + A, lanes containing Maxam-Gilbert G and G + A sequence markers.View Large Image Figure ViewerDownload (PPT)Figure 3Cleavage of the 5′-32P-end-labeledNcoI/Bsu36I DNA fragment (5′-32P-labeled (*) on the nontranscribed (coding) strand at the Bsu36I site) induced by Me-lex at the indicated concentrations in the presence or in the absence of distamycin (dis), by sequential heating at neutral pH and exposure to hot piperidine. Control, undamaged DNA subjected to sequential heating at neutral pH and exposure to hot piperidine; G and G + A, lanes containing Maxam-Gilbert G and G + A sequence markers.View Large Image Figure ViewerDownload (PPT)Figure 2Cleavage of thePshAI/NcoI DNA fragment (5′-32P-labeled (*) on transcribed (noncoding) strand at the NcoI site) of the p53 cDNA, induced by Me-lex at the indicated concentrations in the presence or in the absence of distamycin (dist), by sequential heating at neutral pH and exposure to hot piperidine. Control, undamaged DNA subjected to sequential heating at neutral pH and exposure to hot piperidine; G and G + A, lanes containing Maxam-Gilbert G and G + A sequence markers. The assignment of the cleavage sites was done using gels analyzed under conditions of lower exposure.View Large Image Figure ViewerDownload (PPT) The methylation at A is not random: only A within (or in the vicinity of) A/T-rich lex equilibrium-binding sites (≥3 A/T base pairs) are targeted (Figs. Figure 2, Figure 3, Figure 4, Figure 5). The strongest adduction sites in the restriction fragments are at A398, A402, A403, A441, A445 (Fig.2), A601, A602, A616, A617, A635, and A636 (Fig.3), A598, A583, A553 (Fig. 4), and A982 (Fig. 5). In addition to the preference for A/T-rich regions, there is a strong preference for specific A's within the binding sites. For example, the bands at A635 and A636 are of equal intensity, while the two adjacent A's (A633 and A634) are not methylated at all. Plasmid pLS76 was damaged in vitro with increasing Me-lex concentrations (TableII). Damaged or undamaged plasmids were transfected into yIG397. Transformants were selected on plates lacking leucine but containing sufficient adenine for adenine auxotrophs to grow and turn red. Survival showed a Me-lex concentration-dependent decrease while the mutant frequency increased in a concentration dependent way (Table II). Only 5 mm induced mutants were purified for the molecular analysis as the level of induction (47-fold above background) guaranteed that 97% of the mutants were actually drug-induced.Table IISurvival and mutation induction in undamaged and Mc-lex damaged pLS76 after passage through yIG397 strainMe-lexSurvivalp53 mutant frequencyF aF, mutant frequency of Me-lex damaged/mutant frequency of undamaged DNA.mm%01001.8 × 10−41(n = 6)bn, number of independent transfections,i.e. number of transfections performed with DNA from different damaging treatments.(6/32,601)0.557 ± 223.3 × 10−41.8(n = 10)bn, number of independent transfections,i.e. number of transfections performed with DNA from different damaging treatments.(15/45,669)229 ± 622 × 10−412(n = 4)bn, number of independent transfections,i.e. number of transfections performed with DNA from different damaging treatments.(36/16,366)414 ± 460 × 10−433(n = 5)bn, number of independent transfections,i.e. number of transfections performed with DNA from different damaging treatments.(99/16,466)58.5 ± 6.384 × 10−447(n = 9)bn, number of independent transfections,i.e. number of transfections performed with DNA from different damaging treatments.(121/14,397)a F, mutant frequency of Me-lex damaged/mutant frequency of undamaged DNA.b n, number of independent transfections,i.e. number of transfections performed with DNA from different damaging treatments. Open table in a new tab After purification by successive platings, 90 Me-lex-induced mutants were cultured for plasmid recovery. Twenty-eight mutant clones were negative for p53 cDNA amplification. To determine whether adenine auxotrophy was due to p53 mutations rather than, for example, mutations in the promoter, the p53 open reading frame from nucleotides 125 to 1122 was PCR amplified from the remaining 62 ade− leu+ clones and tested by gap repair. Unpurified PCR product and HindIII-StuI linearized pRDI-22 (16Inga A. Iannone R. Monti P. Molina F. Bolognesi M. Abbondandolo A. Iggo R. Fronza G. Oncogene. 1997; 14: 1307-1313Crossref PubMed Scopus (39) Google Scholar) were co-transfected by electroporation into yIG397. The HindIII-StuI digested pRDI- 22 gapped plasmid has two regions homologous to the terminal regions of the PCR product. After co-transfection and followed by homologous recombination in vivo (gap repair) a p53 expression vector is reconstituted having the wild-type promoter region (derived from pRDI-22) and the core region of the p53 open reading frame (derived from the PCR product). If the clone was initially ade− due to a mutation in the promoter region, the gap repaired transformants will originate almost exclusively white normal size colonies on limiting adenine plates (no small red colonies, gap repair negative), since the promoter in the gap-repaired plasmid, derived from pRDI-22, is wild-type. On the contrary, in case the PCR product contains a single mutation, approximately 100% of the derived transformed clones will give rise to small red colonies (gap repair positive) (see Fig. 1 in Ref. 16Inga A. Iannone R. Monti P. Molina F. Bolognesi M. Abbondandolo A. Iggo R. Fronza G. Oncogene. 1997; 14: 1307-1313Crossref PubMed Scopus (39) Google Scholar). PCR fragments from 27 clones gave a low percentage of red colonies, suggesting that the mutation causing adenine auxotrophy lay outside the region tested by gap repair. The remaining 35 gave 100% red colonies after gap repair, and p53 mutations were found by DNA sequencing in every case. However, two mutants could not be considered independent because both derived from the same damaging treatment and showed the same mutation. Only one of these two mutants was considered for further analysis. All of the remaining 34 independent mutants but one (X272) contained a single mutation (TableIII).Table IIIMe-lex induced mutation spectra at the p53 cDNA after in vitro treatment of plasmid pLS76 and passage into yIG397 cellsMutant n
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