Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide–DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene

Moon-shong Tang2, Jessica B. Zheng, Mikhail F. Denissenko1, Gerd P. Pfeifer1 and Yi Zheng

Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park, Smithville, TX 78957, USA and
1 Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA


    Abstract
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 Abstract
 Introduction
 References
 
We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (±)anti-7ß, 8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers. Given the far reaching ramifications of such conclusions for cancer etiology, a legitimate question is raised regarding the reliability of using the UvrABC incision method for quantifying and determining the sequence-dependency of adduct formation. Is the higher frequency of UvrABC cutting at methylated versus unmethylated CpG sites due to the preference of the nuclease for cutting at those sites or due to the preferential formation of BPDE adducts at those sites? In order to distinguish between these two possibilities, we have analyzed the kinetics of UvrABC incision at BPDE adducts formed at either methylated CpG sites versus other sequences, or unmethylated CpG sites versus other sequences in exon 5 of the p53 gene. We have found that the UvrABC cutting kinetics are identical for both cases. On the basis of these results we conclude that under proper cutting conditions, UvrABC nuclease reacts with and incises with equal efficiency, BPDE adducts formed at methylated or unmethylated CpG sites as well as other sequences, and that the extent of UvrABC incision accurately reflects the extent of BPDE–DNA adduct formation. These conclusions were further supported by results obtained using a DNA synthesis blockage assay.

Abbreviations: BPDE, (±)anti-7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetra-hydrobenzo[a]pyrene; DTT, dithiothreitol; LMPCR, ligation-mediated polymerase chain reaction.


    Introduction
 Top
 Abstract
 Introduction
 References
 
The mutational spectrum and signature mutations in the tumor suppressor p53 gene in human cancers have revealed a wealth of information regarding the etiology of cancer (13). This information is important for cancer prevention and potentially useful for cancer treatment. Interestingly, although >200 sites of mutation have been identified in the p53 gene, 30% of total mutations are located at codons 156, 157, 175, 245, 248, 249, 273 and 282, all of which (except codon 249) contain a guanine in a CpG sequence, and it appears that different cancers often share certain common mutational hotspots. There are certain mutational hotspots which appear to be tissue specific; for example, hotspot mutations at 175, 248 and 273 are observed in ovarian, brain, breast and stomach cancers and leukemia/lymphoma. On the other hand, prostate cancers show only one hotspot mutation at codon 273, and hepatoma cases from the East Asia and sub-Sahara Africa areas have a single hotspot mutation at codon 249 (13). Cigarette smoking-related lung cancers, while having mutational hotspots at codons 248, 273 and 282 that are commonly observed in other cancers, also have one hotspot mutation at codon 157 that is not a mutational hotspot in other cancers (1,2,46). The crystal structure of the p53 protein has revealed that the amino acids encoded by codons 175, 248, 273 and 282 are important for DNA contact and/or pivotal for the integrity of the p53 protein structure (6,7). However, it is not clear whether mutations at these particular mutational hotspot codons contribute more effectively to either loss of tumor suppressor function or gain of oncogenic function than mutations in other codon sequences.

While it is possible that the specific effects of different mutations on p53 gene function, either on its ability to suppress cancer formation or in activating cancer formation, may be tissue or cell dependent, it is also possible that the p53 mutational hotspots in different cancers may result from different carcinogen exposures in different tissues and/or that p53 genes from different tissues have different susceptibilities for carcinogen binding (8). In order to test the latter possibility, we recently mapped the adduct distribution along the p53 gene in normal human bronchial epithelial cells treated with the activated cigarette smoke component, (±)anti-7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE), by using the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques. We found that UvrABC nuclease incisions preferentially occurred at sites corresponding to the p53 mutational hotspots observed in human lung cancers (4). Based on these results, and since we had shown previously that under our conditions UvrABC nuclease incises BPDE–DNA adducts specifically and quantitatively, we concluded that BPDE adducts preferentially form at p53 mutational hotspot sites in lung cancers (4,9). Our subsequent studies have confirmed that: (i) all these preferred sites for BPDE binding are guanine residues within CpG dinucleotides (10,11); (ii) methylation of cytosine at such sites is the determining factor for preferential binding (10,11); and (iii) repair of BPDE adducts formed at these sites is significantly slower than at other sequences (11). These results not only provide a molecular link between a known carcinogen and lung cancer but also lead us to hypothesize that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantages, that is the basis of the p53 mutation patterns seen in human cancers (8). These results also suggest that an epigenetic factor, cytosine methylation at CpG sites, may play a significant role in determining cancer susceptibility (8).

Obviously, the methodology used to obtain these results merits careful evaluation, particularly with regard to the question of whether UvrABC incision represents the sequence-dependent adduct formation or whether it merely reflects the sequence-dependent UvrABC incision preference. Does BPDE preferentially bind guanines at methylated CpG sites or does UvrABC preferentially incise BPDE–guanine adducts formed at methylated CpG sites?

In order to resolve this issue, we have used analyses of the UvrABC incision kinetics of adducts formed at different sequences to assess whether there are significant sequence-dependent and/or methylation-dependent adduct cutting preferences by this enzyme system. We also further investigated the sequence dependence versus methylation dependence of adduct formation by determining the relative effectiveness of blockage of Sequenase-mediated DNA synthesis on BPDE-modified CpG-methylated versus unmethylated DNA templates.

Although UvrABC nuclease is able to incise BPDE adducts formed in plasmid DNA quantitatively (9), it had not previously been determined whether this enzyme system would show differences in the rate constant of cutting of adducts formed at different sequences, or at methylated versus unmethylated CpG sites. Although DNA fragments with a site-specific adduct might represent ideal substrates for determining the role of DNA sequence or cytosine methylation at CpG sites on an adduct's susceptibility for UvrABC cutting, the number of sequences that would need to be tested makes it unfeasible to take this approach. Furthermore, both the relatively short lengths of oligonucleotides containing site-directed adducts [which, for practical reasons, are generally limited to ~60 bp (12,13)] and the purity of the constructs may adversely affect the UvrABC cutting efficiency which consequently obscures the determination of the DNA sequence effect per se on adduct formation and adduct–enzyme affinity.

For these reasons, we used 259 bp DNA fragments PCR amplified from exon 5 of the p53 gene. These DNA fragments were 5'-32P-labeled by using one-end labeled primer and methylated at CpG sites by CpG methylase (8,10,11). Methylated and unmethylated DNA fragments were subsequently modified with the same concentration of BPDE under conditions which produced an average of less than one adduct per DNA fragment. The DNA fragments were then treated with UvrABC under conditions in which the enzyme:DNA molar ratio was >=6.

It is worth noting that there are no DNA polymerases and ligases present under our reaction conditions and, therefore, the results of UvrABC incision should be irreversible. We previously have demonstrated that C5 cytosine methylation results in much stronger UvrABC incision bands at CpG sites in BPDE-modified DNA; the extent of enhancement appears to be sequence dependent, and varies from 2- to 10-fold (8). If this enhancement of UvrABC cutting at methylated CpG sites is due to the enzymes having higher cutting efficiency towards BPDE adducts formed at methylated CpG sites versus unmethylated CpG or other sequences, then the kinetics of UvrABC incision should be different for methylated CpG sites and unmethylated CpG sites or other sequences. To test this possibility, the time course of UvrABC cutting for each of these different substrates was determined. A typical result is shown in Figure 1Go, demonstrating that UvrABC incision at all BPDE-binding sites, in both methylated and unmethylated DNA, is a function of incubation time and appears to plateau after 30 min of incubation.



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Fig. 1. UvrABC incision time course on methylated and unmethylated DNA modified with BPDE. Methylated and unmethylated 5'-32P-labeled 259 bp fragments of exon 5 of the p53 gene were modified with the same concentration of BPDE (5x10–5 mg/ml) as described previously (8) and then reacted with UvrABC for different lengths of time. The reactions were stopped by phenol and ether extractions followed by ethanol precipitation. The resultant DNAs were separated by denaturing 8% PAGE. Lanes 1 and 5–11, methylated DNA; lanes 2–4 and 12–18, unmethylated DNA. Meth, CpG methylation; +, with CpG methylation; –, without CpG methylation. Codons containing a guanine in a CpG site are indicated with an asterisk at the right of the panel. C* marks the methylated cytosine at CpG sites indicated by a missing band in the C-specific reaction. G, GA and TC represent Maxam–Gilbert sequencing reactions. UvrABC nuclease makes dual incisions of seven bases 5' to, and four bases 3' to, a BPDE–guanine adduct. Therefore, the position of the UvrABC incision band corresponding to the adducted guanine is seven bases lower than the position of that guanine in the Maxam–Gilbert guanine ladder. The 259 bp fragments were obtained by amplification (20 cycles) of exon 5 region of the p53 gene from p53 gene containing plasmid (pAT153P53{pi} obtained from L.Crawford and S.P.Tuck, Imperial Cancer Research Fund Laboratories, London, UK). The plasmids were hybridized with two oligonucleotide primers 5'-TGCC- CTGCTTTCAACTCTGTCTCC-3' and 5'-CCAGCCCTGTCGTCTCTC- CAGCC-3' with the former labeled at the 5' end with [{gamma}-32P]ATP according to the method described previously (8). The 32P single-end labeled DNA amplified fragments (259 bp) were purified through 8% PAGE. The 32P single-end labeled DNA fragments were subjected to SssI methylase treatment with S-adenosylmethionine to methylate all cytosines at CpG sites according to vendor's instructions (8). The UvrABC nuclease reactions were carried out in a total volume of 25 µl containing 50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 1 mM ATP, 100 mM KCl, 1 mM dithiothreitol (DTT), 15 nM UvrA, 15 nM UvrB, 15 nM UvrC and substrate DNA (2 nM). UvrA, UvrB and UvrC proteins were isolated from the Escherichia coli K12 strain CH296 (recA, endA/F'lacIQ) carrying plasmids pUNC45 (uvrA), pUNC211 (uvrB), pDR3274 (uvrC) (18). The proteins were purified as described previously (19).

 
Eighteen well-resolved bands representing UvrABC cutting within the middle region of the DNA fragment, including eight CpG sites and 10 non-CpG sites, were quantified. Since it has been found that efficient UvrABC cutting requires a minimal DNA length, no attempt was made to quantify DNA adducts formed near the ends of the DNA fragments (14). Figure 2Go shows the UvrABC cutting time courses for each of the 18 sites. These results demonstrate that 90% of UvrABC cutting occurs during the first 30 min, and that the cutting kinetics for both methylated CpG sites versus other sequences, and unmethylated CpG sites versus other sequences are nearly identical, although the final extent of cutting in methylated CpG sites is higher than at their unmethylated counterparts (Figure 3Go).




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Fig. 2. Kinetics of UvrABC incision on methylated and unmethylated DNA modified with BPDE. Eighteen well-separated bands in Figure 1Go were quantified using Bio-Image Open Windows (Version 3 System) with a Howtek Scanmaster 3+ and whole band analysis software. The band intensities were adjusted by the amount of sample applied onto the gel, and then normalized to the band intensities at 60 min incubation. (A) Quantification from methylated DNA; (B) quantification from unmethylated DNA. The sequences of the codons are described in the right of the panel. The asterisk represents the guanine residue modified with BPDE. The lines in the panel represent the mean value of the different sequences (right of the panel); for clarity, only four sequence points are presented in the panel and the rest of the sequence points are within the length of the bar.

 


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Fig. 3. The effects of cytosine methylation at CpG sites on BPDE–DNA adduct formation. Twenty-four well-separated bands in lanes 10 and 17 of Figure 1Go were quantified and normalized to the amount of 32P applied to the gel. The y-axis represents the relative intensity of UvrABC cutting of methylated DNA (top) and unmethylated DNA (bottom). The codons with a guanine at CpG sites are indicated.

 
The relationship between UvrABC cutting and BPDE–DNA adduct formation can be summarized as Pn = Kn·E·Sn, where Sn represents the DNA adducts formed at any particular sequence, Pn represents the resulting UvrABC cutting at this sequence, and E represents the molar concentration of UvrABC.

There are two possible explanations for the different Pn that we have observed at different sequences, and at methylated versus unmethylated CpG sites; either Kn or Sn must be sequence dependent and C5 cytosine methylation dependent. If Kn is sequence/methylation dependent, one would expect that after long incubation times, Pn would eventually be equal for methylated, unmethylated CpG sites and other sequences, since in our reaction conditions E > S. However, results in Figure 1Go show that: (i) after 30 min of incubation, UvrABC cutting (P) for all sequences reached plateaus and (ii) even after further incubation (90 min) the different extents of UvrABC cutting for different sequences in both methylated and unmethylated DNA remain the same as those observed at 30 min of incubation. Furthermore, the rate constants of UvrABC cutting for methylated and unmethylated CpG sites and other sequences are identical (Figure 2Go). These results rule out the possibility that Kn is sequence and C5 methylation dependent. Therefore, the different values of Pn for methylated versus unmethylated CpG and for different sequences must reflect differences in adduct formation; Sn must be C5 methylation and sequence dependent.

On the basis of these results we conclude that (i) the degree of UvrABC incision (P) represents the extent of BPDE–DNA adduct formation and (ii) enhancement of UvrABC cutting at C5 methylated CpG sites is due to the preferential binding of BPDE at methylated CpG sites, not due to more efficient cutting by UvrABC at BPDE–DNA adducts formed at methylated CpG sites.

To further strengthen these two conclusions, we have used a DNA replication stoppage assay to demonstrate the effect of methylation at CpG sites on BPDE–DNA binding. It has been demonstrated that BPDE-modified guanines in the DNA template strand block the progression of DNA chain elongation (1517). If BPDE binds more efficiently at guanines within methylated CpG sites than to guanines at unmethylated CpG sites or other sequences, then this different extent of BPDE binding should also be reflected by the extent of DNA synthesis blockage. Plasmid DNAs with a p53 gene insert were treated with and without CpG methylase, and then modified with BPDE. These BPDE modified plasmid DNAs were used as templates for determining Sequenase-mediated DNA synthesis at an exon 8 region using 5'-32P-labeled primers. The results in Figure 4Go show that CpG-methylated DNA templates resulted in significantly more DNA synthesis blockages at CpG sites (codons 273, 282, 283, 290 and 298) than observed with unmethylated DNA templates. These results are consistent with our previous finding that CpG methylation in exon 8 DNA fragments increases BPDE binding at CpG sites in codons 273, 282, 283 and 290 (8).



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Fig. 4. Determination of the position and the degree of DNA synthesis blockage using BPDE modified DNA templates. Plasmid pAT153P53{pi} was methylated to completion with SssI DNA methylase or mock treated as described (8). The plasmids were linearized with EcoRI, phenol extracted and ethanol precipitated. Both methylated and unmethylated plasmids were modified with BPDE at final concentrations of 0.5 or 10 mM in the dark for 90 min. Unreacted BPDE was removed by repeated diethyl ether and isoamylalcohol extractions, and the DNA was ethanol precipitated. A p53-specific primer (5'-AAGAGGCAAGGAAAGGTGATA) was 5'-32P-labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP. BPDE-modified or unmodified plasmid (3 µg) was mixed with the 32P-labeled primer (0.5 pmol) in 40 mM Tris–HCl pH 7.7, 50 mM NaCl in a volume of 15 µl, heated to 95°C for 5 min, and then the primer was annealed at 48°C for 30 min. Nine microliters of 20 mM DTT, 20 mM MgCl2 and 0.25 mM dNTPs was added together with 5 U of Sequenase 2.0 and the reaction was incubated at 48°C for 20 min. Sequenase was heat inactivated at 67°C for 15 min, the DNA was ethanol precipitated, denatured in formamide loading buffer and loaded onto an 8% polyacrylamide gel. The gel was dried and exposed to X-ray film. The position of the guanine residue in the sequence was identified by Maxam–Gilbert sequencing. The codon number is depicted on the right and the asterisk indicates the CpG site.

 
It is possible that UvrABC cutting may show some small degree of sequence-dependent preference. However, under the conditions in which the enzyme–DNA adduct molar ratio is excessive and in which the period of incubation time allows the completion of >90% of cutting, such a small sequence-dependent preference of UvrABC cutting should not affect the validity of using enzyme cutting for adduct quantitation at the sequence level.


    Acknowledgments
 
We thank Dr Gerry Adair and Miss Yen-Yee Tang for critical review of this manuscript. This research was supported by grants ES03124, ES08389 (M.-s.T.), CA65652 and CA69449 (G.P.P.) from the National Institutes of Health.


    Notes
 
2 To whom correspondence should be addressed Email: sa83103{at}odin.mdacc.tmc.edu Back


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Received September 15, 1998; revised February 18, 1999; accepted March 1, 1999.