Mutagenesis, Vol. 16, No. 1, 65-69,
January 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide–deoxyadenosine adducts into M13mp7L2
1 Chemistry of Carcinogenesis Laboratory, National Cancer Institute–FCRDC, Frederick, MD 21702 and 2 Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Abstract |
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The adduct that would arise from cis opening of (+)-(1S,2R,3R,4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the underlined site into four oligonucleotides, 5'-CAGATTTAGAGTCTGC-3', 5'-CAGTGCAGATTTAGAG-3', 5'-GTGCAGATTTAGA-3' and 5'-TGCAGATTTA-3'. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the underlined A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40–58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.
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Introduction |
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Interaction between metabolites of chemical carcinogens, such as polycyclic aromatic hydrocarbons, and DNA can cause mutation in oncogenes and tumor suppressor genes which is believed to initiate the carcinogenic process (Peters and Vousden, 1997
). Several addition products (adducts) are often detected in the reaction of these reactive metabolites and DNA, so site-specific mutation assays have been developed in order to assess a specific adduct's activity in the mutagenic process (Singer and Essigmann, 1991; Loechler, 1996). Site-specific mutation studies of polycyclic aromatic hydrocarbon diol epoxide–DNA adducts have been performed in double-stranded (Mackay et al., 1992; Jelinsky et al., 1995; Shukla et al., 1997a,b; Lavrukhin and Lloyd, 1998) and single-stranded (Chary et al., 1995; Moriya et al., 1996; Min et al., 1996; Hanrahan et al., 1997; McNees et al., 1997; Fernandes et al., 1998; Page et al., 1998, 1999; Pontén et al., 1999) DNA.
In previous work (Page et al., 1998, 1999; Pontén et al., 1999, 2000), investigating the mutational consequences of replication of M13mp7L2 constructs containing benzo[a]pyrene or benzo[c]phenanthrene diol epoxide (B[c]PhDE) adducts (see Figure 1 for a schematic overview of the experimental procedure), we have noted that a fraction (sometimes a large fraction) of plaques do not align with any probes when screening by differential hybridization. We have sequenced a number of these plaques to analyze their properties and found that the majority lacked the oligonucleotide insert. In a few cases, the hairpin that should have been cut out by the restriction enzyme, EcoRI, was still present. The high number of plaques lacking the insert has proved to be a considerable inconvenience in screening by differential hybridization. In some cases, the majority of the plaques could not be assigned. This vastly increased the number of plates that had to be screened in order to obtain reliable numbers for comparing mutation frequencies from different adducts in different contexts. We were interested in investigating the reason for the failure to insert the oligonucleotide into the constructed vectors. Therefore, we constructed oligonucleotides with sequences based on context II(A), 5'-CAGATTTAGAGTCTGC-3' (Pontén et al., 1999) containing an adduct, (A), cis B[c]PhDE-2/S (Figure 2) that we had examined previously. This particular S-adducted oligonucleotide was chosen as a starting point, since it corresponded to the highest mutational frequency of the eight isomeric dAdo adducts previously observed by us in two different sequences (Pontén et al., 2000). In the present experiments, variants of differing length were constructed (Figure 2), all of which had the adduct in the middle of the sequence. The sequence context around the adduct was maintained by repeating a portion of the linearized M13 sequence (at its 3'-end) on the 5'-end of the original context and cutting the 3'-end of the insert by the same number of bases. Mutation frequencies and distributions were similar for all four constructs, indicating that changes of the partial M13 sequence six or more bases 5' to the adduct or five or more bases 3' to the adduct did not significantly alter its mutagenic properties. Moving the adduct from the end of the sequence to the middle markedly improved the efficiency of ligation, and reduced the number of plaques lacking the insert by almost 50%. However, the length of the insert had little effect.
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Materials and methods |
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Oligonucleotide synthesis
Oligonucleotides were prepared by semi-automated synthesis as described (Lakshman et al., 1992
; Pontén et al., 1999) on a 2 µmol scale using a diastereomeric mixture of 5'-dimethoxytrityl-3'-(N,N-diisopropyl)(ß-cyanoethyl)phosphoramidites corresponding to the cis-opened N6-dAdo adduct of (±)-B[c]PhDE-2 (Pilcher et al., 1998; Ponten et al., 1999). Yields on coupling of the modified phosphoramidites, estimated from the release of dimethoxytrityl alcohol on detritylation immediately after the manual coupling step, ranged from 10% to 50%. The diastereomeric, adducted oligonucleotides were purified by high performance liquid chromatography (HPLC) (Table I) after removal of the 5'-dimethoxytrityl protecting group. Configurations of the adducts were assigned after enzymatic hydrolysis to nucleosides (Sayer et al., 1991; Pontén et al., 1999) of one member of each diastereomeric pair of oligomers: the early eluting 13-mer [context II(A)-2] and the late eluting 10- [context II(A)-3] and 16-mers [context II(A)-1]. Comparison of the circular dichroism (CD) spectra of the nucleoside adducts with those of the known dAdo adducts from B[c]PhDEs (positive at ~250 nm and negative at ~280 nm for the S adducts (Agarwal et al., 1987) indicated that in all three cases, the late-eluting diastereomer had the desired S absolute configuration.
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CD spectra of the single-stranded oligonucleotides (two diastereomers of each sequence) are shown in Figure 3
. These CD spectra, like those of the adducted 16-mer sequence II(A) (Pontén et al., 1999), containing cis-opened B[c]Ph DE-2 dAdo adducts, are weak and are not independently diagnostic of absolute configuration. The CD spectra of the present oligonucleotide adducts exhibited some similarities to those of the corresponding 16-mer sequences previously reported [context II(A)] with a R- or S-adducted dAdo at position 4 from the 5'-end. Thus, the R isomers show only positive bands above ~230 nm whereas the S diastereomers have a distinct negative band around 255 nm, a positive band at ~270 nm and two weak, negative bands above 280 nm. These bands for the S adduct were present but less pronounced in the corresponding context II(A) 16-mer containing cis B[c]PhDE-2/S at position 4.
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Construction of vector and mutagenesis assay
Construction of the modified vector and its use in the mutagenesis assay were as described (Page et al., 1998
; Pontén et al., 1999) (see Figure 1
) essentially following the procedure developed in Lawrence's laboratory (Banerjee et al., 1988; Gibbs and Lawrence, 1993). In brief, the M13mp7L2 DNA vector was linearized using EcoRI (2 units/µg DNA at 30°C for 2.5 h) and purified by phenol extraction. The oligonucleotide (50 pmol) to be inserted was phosphorylated, annealed with a complementary uracil-containing scaffold (2 pmol) with 20-base long overhanging ends complementary to the single-stranded vector. These were then annealed with and ligated (30 units T4 DNA ligase) into M13mp7L2 (1 pmol). Finally, the constructed vector–scaffold complex was treated with uracil DNA glycosylase (1 unit) which removes the uracil bases in the scaffold. The scaffold containing abasic sites was degraded by the intrinsic endonucleases in Escherichia coli after transfection (see below). The constructed vectors were transfected into competent (CaCl2-treated) E.coli SMH77 (20 ng DNA/100 µl cells) that were SOS-induced (40 J/m2 UV light; Gibbs and Lawrence, 1993). Top agarose containing 0.03% 5-bromo-4-chloro-3-indolylgalactoside (X-gal; Amersham Inc.) and 0.3 mM isopropylthiogalactoside (IPTG; Amersham Inc.) was added, the mixture was poured on to agar plates and the bacteria were grown overnight at 37°C. X-Gal is cleaved by active ß-galactosidase to produce deep blue dibromodichloroindigo; IPTG is added as an inducer of the ß-galactosidase operon. Upon induction by IPTG, a defective ß-galactosidase is synthesized, which is complemented by the ß-galactosidase fragment produced by the phage (Messing, 1983). In M13mp7L2, the reading frame for the ß-galactosidase mRNA has a 2 base frameshift, so insertion of a 16-, 13- or 10-mer puts it back in frame. Plaques containing M13mp7L2 phage without the oligonucleotide insert will be colorless in the presence of X-gal and IPTG, whereas plaques formed by phage containing the inserted oligonucleotide will be deep blue in color. Ligation efficiencies were determined using Southern hybridization and by quantifying the radiolabeled bands corresponding to circular and linear M13 (Page et al., 1998; Pontén et al., 1999). In most cases, 30 blue plaques (i.e. containing the insert) from each construct were picked randomly for sequencing using an ABI-PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer; see Page et al., 1998; Pontén et al., 1999). |
Results and discussion |
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Adducted oligonucleotides were synthesized from the diastereomeric phosphoramidite (Pilcher et al., 1998
; Pontén et al., 1999) corresponding to the cis-opened dAdo adduct derived from (±)-B[c]PhDE-2. After enzymatic hydrolysis of one member of each pair, configuration at the point of attachment to the adenine base was assigned on the basis of the CD spectra of the derived nucleoside adducts, whose absolute configuration had previously been established (Agarwal et al., 1987). Although our synthetic procedure gives both R- and S-adducted oligonucleotides, only the oligonucleotides containing the S adducts were used in the present experiments, since these adducts gave rise to the highest mutational frequency (Pontén et al., 2000) in context II(A). After chromatographic purification (Table I), the newly synthesized oligonucleotides were homogeneous by analytical HPLC. The three newly synthesized oligonucleotides were further purified by gel electrophoresis to remove any possible contamination by shorter, adduct-containing `failure' sequences, which might not be well separated chromatographically from the desired oligomers. After this final purification step, aliquots of the four S-adducted oligonucleotides [context II(A) and each of the three newly synthesized sequences, Figure 2], as well as their four unsubstituted control sequences, were end-labeled and subjected to electrophoresis (not shown), which demonstrated that they were free from any contaminating sequences of different lengths.
The apparent ligation efficiencies were in the range 24–45% calculated as the amount of circular DNA divided by the sum of the amounts of linear and circular DNA (Table II, see below). For cis B[c]PhDE-2/S-adducted context II(A), where the adduct is located at the fourth base from the 5'-end of the oligonucleotide, the presence of adduct reduced the ligation efficiency as compared with the unsubstituted control (83% and 75% of control; Table II). In contrast, the ligation efficiencies for all sequences where the adduct was located in the middle [contexts II(A)-1 to -3], were essentially unaltered or slightly higher than the control, regardless of the length of the inserted oligonucleotide.
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The numbers of plaques screened were in the range 1791–2581 for the unsubstituted controls and 357–768 for the cis B[c]PhDE adducted vectors (Figure 4
). Three separate experiments were done with several independent constructs. Some variation in the efficiency of transfection was noted, but variations in the survival (see below) and number of colorless plaques were minor. The unsubstituted constructs resulted in 2–11% and the adducted sequences in 23–53% colorless plaques (Figure 4). The majority of the colorless plaques probably result from rejoining of the blunt ends of linear M13 [as shown by sequencing of colorless plaques from other sequences (unpublished); see Figure 1]. We have previously changed our protocol by annealing the oligonucleotide and scaffold first before adding M13 (rather than annealing scaffold and M13 before adding oligonucleotide), to minimize the risk of hairpin formation by the scaffold, which could result in rejoining of the cut M13 ends. A few of the colorless plaques might arise from uncut M13 [as shown by sequencing of colorless plaques from other sequences (unpublished)] and, possibly, some may contain the insert and additionally have untargeted mutations resulting in a stop codon for ß-galactosidase or deletions resulting in frameshifts. The original adducted sequence [context II(A)] resulted in 43–53% colorless plaques whereas adducted contexts II(A)-1 to -3 resulted in 23–34% colorless plaques (Figure 4). Thus, moving the adduct from the end of the sequence to the middle reduced the number of colorless plaques by almost 50%.
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Survival was calculated as the number of blue plaques resulting from transformation with adducted, compared with the corresponding unsubstituted constructs, where the latter were considered to be 100%. The survival of cis B[c]PhDE-2/S in context II(A) (11.3%) was consistent with previous results (13%; Pontén et al., 1999), whereas the survival in contexts II(A)-1 to -3, where the adduct was located in the middle of the insert, was about twice as high, regardless of the length of the original oligonucleotide (see legend to Figure 4
). We conclude from these results that the length of the adducted oligonucleotide does not affect the ligation efficiency to any major extent, since contexts II(A)-1 to -3 all gave approximately the same ligation efficiencies. The difference between the 16-mer [context II(A)] and contexts II(A)-1 (also a 16-mer), -2 and -3 is likely to be explained by a difference in the ability of the scaffolds to anneal with themselves in preference to the adducted oligonucleotide. The resulting hairpins could loop out, making it possible for the ligase to rejoin the blunt ends of the M13. Despite the attempt to avoid this by annealing the scaffold with an excess of the oligonucleotide insert (25-fold), some distortion at the adduct site may result in weaker interaction with the scaffold, so that hairpin formation by the scaffold is energetically possible, even in the presence of excess insert. Context II(A) has only three bases 5' to the adduct, whereas contexts II(A)-1 to -3 have eight, six and five bases 5' to the adduct, respectively. Since there was no noticeable difference between the latter contexts, five or more bases 5' to the adduct may be adequate to hold the end annealed to the scaffold. There is also a difference in the possible self-annealing of the insert, with several more bases in context II(A) able to self-anneal. Despite marked improvement on using the new inserts II(A)-1 to -3, the scaffold may still anneal suboptimally to these adducted sequences. Thus, even with these new, adducted inserts, ~25% colorless plaques (due to circularization of the phage without incorporating the insert) were still observed with these oligonucleotides upon transfection and replication (see below). Consequently, the ligation efficiency of all the adducted constructs may be somewhat overestimated when based on total circular DNA.
It should also be pointed out that survival of the adducted phage is underestimated by comparing numbers of blue plaques obtained with the adducted inserts relative to the controls, since less insert-containing phage was generally present on the plates resulting from transfection with the adducted DNA, although the same total amount of DNA was added per plate. This is especially notable in the case of the adducted context II(A), where about half of the obtained plaques lacked the insert. The total amount of insert-containing DNA that results in blue plaques depends on both the (unknown) efficiency of ligation of the insert into the M13 DNA and the subsequent viability on replication of the phage containing the adduct. It is not possible to estimate the relative contributions of these two factors from the present data.
Table III summarizes the number of mutants found in the different constructs. None of the sequenced plaques from unsubstituted constructs contained any mutants. Since only 30 plaques from each were sequenced, the mutation frequency in these controls was thus <3.2%. For the adducted constructs, very high mutation frequencies were obtained (40–58.6%). In agreement with previous results, the preponderance of the mutational events were A
G base substitutions, and the rest of the mutations were AT. For three of the contexts [contexts II(A), II(A)-1 and II(A)-3] there was no significant difference between the mutation frequencies. A lower mutation frequency was noted for context II(A)-2, but this may possibly be in error because of the small number of sequenced plaques.
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The present findings demonstrate that our previous mutation results for this adduct in context II(A) are independent of the length of the inserted DNA fragment; most importantly, they show that sequence alterations (repetitions of two to five bases of the M13 DNA on the 5'-side of the adduct) at a position six or more bases away from the adduct have little or no effect on its mutagenic consequences. Thus, so long as the local sequence surrounding the adduct is maintained, the size of the insert and the position of the adduct in it could, in principle, be altered to improve ligation efficiency while still permitting valid comparisons with our previous, extensive data, all of which have been obtained using 16-mers containing adducts four bases from the 5'-end.
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Acknowledgments |
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We thank Dr Christopher Lawrence for M13mp7L2 and E.coli SMH77, Dr Gary Pauly and Dr Monica Cooper for valuable discussions during the preparation of the manuscript and Dr Hye-Young Kim for assistance in the purification and enzymatic hydrolyses of the adducted oligonucleotides.
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Notes |
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3 To whom correspondence should be addressed. Tel: +1 301 846 5839; Fax: +1 301 846 6146; Email: ponteni{at}ncifcrf.gov
Deceased May 26, 1999. |
References |
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Received on June 1, 2000; accepted on August 21, 2000.
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