Slow Repair of Bulky DNA Adducts Along the Nontranscribed Strand of the Human P53 Gene May Explain the Strand Bias of Transversion Mutations in Cancers

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers

Mikhail F Denissenko1, Annie Pao2, Gerd P Pfeifer1 and Moon-shong Tang2

1Beckman Research Institute, City of Hope, Duarte, California 91010; 2University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA

Using UvrABC incision in combination with ligation- cancer development (Hollstein et al., 1991, 1996; Jones mediated PCR (LMPCR) we have previously shown that et al., 1991; Harris and Hollstein, 1993; Greenblatt et benzo(a)pyrene diol epoxide (BPDE) adduct formation al., 1994; Harris, 1996; Ruddon, 1995). along the nontranscribed strand of the human p53 gene is The p53 mutations found in human cancers are of highly selective; the preferential binding sites coincide various types, and occur at dierent sequences with the major mutation hotspots found in human lung (Greenblatt et al., 1994; Hollstein et al., 1996). More cancers. Both sequence-dependent adduct formation and than 200 mutated codons of the p53 gene have been repair may contribute to these mutation hotspots in identi®ed in various human cancers, and the types of tumor tissues. To test this possibility, we have extended mutations often seem to bear the signature of the our previous studies by mapping the BPDE adduct etiological agent (Greenblatt et al., 1994; Hollstein et distribution in the transcribed strand of the p53 gene and al., 1996). One of the most notable examples is lung quantifying the rates of repair for individual damaged cancer. In lung cancers from cigarette smokers, 40% of bases in exons 5, 7, and 8 for both DNA strands of this p53 gene mutations are G to T transversions and 24% gene in normal human ®broblasts. We found that: (i) on are G to A transitions, while in lung cancers from both strands, BPDE adducts preferentially form at CpG nonsmokers, only 13% of mutations in this gene are G sequences, and (ii) repair of BPDE adducts in the to T transversions and more than 60% are G to A transcribed DNA strand is consistently faster than repair transitions (Greenblatt et al., 1994; Hollstein et al., of adducts in the nontranscribed strand, while repair at 1996). Furthermore, 90% of the G to T transversion the major damage hotspots (guanines at codons 157, 248 mutations in the p53 gene in lung cancers occur on the and 273) in the nontranscribed strand is two to four nontranscribed strand. It has been well established that times slower than repair at other damage sites. These most bulky carcinogen-induced mutations are G to T results strongly suggest that both preferential adduct transversions, while most spontaneous mutations are G formation and slow repair lead to hotspots for mutations to A transitions (presumably arising by deamination of at codons 157, 248 and 273, and that the strand bias of 5-C methylated cytosine at CpG sites) (Jones et al., bulky adduct repair is primarily responsible for the 1991). In lung cancers of cigarette smokers p53 gene strand bias of G to T transversion mutations observed in mutations are concentrated at codons 157, 179, 245, the p53 gene in human cancers. 248, 273 and 282 (Greenblatt et al., 1994; Hollstein et al., 1996). In contrast, no mutation hotspots are Keywords: BPDE; DNA repair; mutation; p53 observed in lung cancers from nonsmokers, and only a single mutation hotspot at codon 249 is observed in radon-associated lung cancers (Greenblatt et al., 1994; Hollstein et al., 1996). Together, these results strongly Introduction suggest that mutations in the p53 gene in cigarette smokers result from DNA damage from exogenous The tumor suppressor p53 functions as a guardian of agents. the genomic integrity of the cell; it coordinates the Benzo[a]pyrene (BP) is a major carcinogen of cellular responses to DNA damage by inducing either cigarette smoke as well as an environmental pollu- cell cycle arrest to increase the opportunity for repair tant; in vivo, BP is metabolically activated to BPDE, of damaged DNA, or programmed cell death to which binds to nucleophilic DNA at the exocylic amine sacri®ce cells with massive and irreparable DNA of guanine (for a review see, Harvey, 1991). Results damage (Hollstein et al., 1991; Levine et al., 1991; obtained in a number of animal model systems have Harris, 1993; Levine, 1997; Vogelstein and Kinzler, strongly suggested that BPDE induced DNA damage 1992). Mutation of the p53 gene is one of the most initiates carcionogenesis (for review, see Harvey, 1991; common features in neoplastic transformation (Holl- Harvey, 1981; Weinstein, 1981; Pelkonen and Nebert, stein et al., 1991, 1996; Jones et al., 1991; Harris and 1982; Beland and Poirier, 1989). The binding BPDE to Hollstein, 1993; Greenblatt et al., 1994; Harris 1996; DNA is greatly aected by DNA sequence context and Ruddon, 1995). It has been found that more than 50% nucleosome structure (Kaneko and Cerutti, 1982; Boles of human cancers have mutations in the p53 gene with and Hogan, 1984; Osborne, 1990; Tang et al., 1992; these mutations occurring at dierent stages during Smith and MacLeod, 1993; Thrall et al., 1994). Recently, using UvrABC incision and LMPCR methods to map BPDE-DNA adduct distribution in Correspondence: Moon-shong Tang or Gerd Pfeifer the human p53 gene in cultered HeLa cells, normal Received 8 August 1997; accepted 15 October 1997 human ®broblasts and bronchial epithelial cells, we Slow repair of DNA adducts in the human p53 gene MF Denissenko et al 1242 have found that BPDE-guanine adducts form prefer- occur at codons 170 and 290. It is possible that BPDE- entially at codons 157, 248 and 273 (Denissenko et al., adduct-induced mutations that result in amino acid 1996); these preferential binding sites correspond changes at codons 170 and 290 may not aect normal, exactly to the major mutational hotspot sites found wild-type function of the p53 gene product. Indeed, a in smoking-associated human lung cancers (Greenblatt G to T transversion (ACG to ACT) at codon 170 et al., 1994; Hollstein et al., 1996). would lead to a silent mutation. In the present work we have further investigated BPDE-DNA adduct formation and repair in cultured Repair of BPDE adducts formed at mutational hotspots human cells, at single nucleotide resolution, and have is two to four times slower for sites in the nontranscribed extended our understanding of the relationship between strand than sites in the transcribed strand BPDE induced DNA damage, repair and the mutational hotspots and the strand bias of G to T It has been shown that 90% of all G to T transversion transversion mutations in the p53 gene in lung cancer. mutations found in the p53 gene of lung cancers in cigarette smokers occur at G's on the nontranscribed strand of this gene (Greenblatt et al., 1994; Hollstein et Results al., 1996). These results suggest that either BPDE-DNA adduct formation is more ecient in the nontranscribed strand than in the transcribed strand, or removal of BPDE preferentially binds CpG sequences in both the adducts from the nontranscribed strand is much slower. transcribed and nontranscribed strands of the p53 gene While the results in Figures 1 ± 3 show that BPDE binds Previously, using UvrABC-LMPCR methods, we have strongly at individual CpG sites on both the nontran- shown preferential DNA adduct formation by BPDE scribed strand and transcribed strand, the nature of at speci®c sites in codons 157, 248 and 273 of the LMPCR methodology precludes a precise assessment of nontranscribed strand of the human p53 gene; these the relative extent of binding for the two DNA strands. sites correspond to the major mutational hotspots Thus, in order to distinguish these two possibilities, we found in lung cancer (Denissenko et al., 1996). Our have determined the time courses for BPDE-DNA more recent studies have demonstrated that all such adduct repair in both the transcribed and nontran- preferential BPDE binding sites are at CpG sequences scribed strands of the p53 gene in cultured human cells and that cytosine methylation at these sequences treated with 2 mM of BPDE. Results presented in Figures promotes this preferential binding (Denissenko et al., 1 ± 3 clearly demonstrate that at mutational hotspots in 1997). These results suggested that one might also codons 157, 248, 273 and 282, repair of BPDE-DNA expect preferential binding of BPDE at CpG sites in adducts on the nontranscribed strand is signi®cantly the transcribed strand opposite to hotspot binding sites slower than repair of adducts on the transcribed strand. in the nontranscribed strand of the p53 gene, since in While repair of BPDE adducts in the nontranscribed vivo maintenance methylation mechanisms maintain strand is generally slower than in the transcribed strand, symmetrical methylation of cytosines on both DNA much greater sequence-dependent variation in repair strands at such CpG sites (Tornaletti and Pfeifer, eciency is seen on the nontranscribed strand than in the 1995). To test this possibility we have used UvrABC transcribed strand. BPDE adducts on the nontranscribed incision in combination with LPMCR to map the strand in codons 181, 244 and 276 are repaired with a distribution of BPDE adducts along DNA sequences faster rate than those at mutational hotspot sites in encompassing exons 5 ± 8 of the transcribed strand of codons 157, 248, 273 and 282. In contrast, repair of the p53 gene, in cultured human cells treated with 2 mM BPDE adducts in the transcribed strand at these codon of BPDE. Results presented in Figure 1 ± 3 show that positions shows much less sequence-dependence; regard- most CpG sites in the transcribed strand (codons 156, less of the initial eciency of formation, all of the 158, and 170 in Figure 1b, lane 6; codons 244, and 248 adducts on the transcribed strand appear to be rapidly in Figure 2b, lane 6; codons 273, 282, 283, and 290 in and uniformly repaired after 8 h. Figure 3b, lane 6), including those sites opposite to The repair kinetics for BPDE adducts formed at mutational hotspot sites in the nontranscribed strand various sites along both the transcribed and nontran- (codons 157, 158, 245, 248, 273 and 282), are indeed scribed strands of the p53 gene have been quanti®ed preferential BPDE binding sites. It is also worth noting for exons 5, 7 and 8 of the p53 gene. Figure 4 shows, that although guanines on the nontranscribed strand of for each BPDE adduct site the time period required for codons 158, 245, and 282 have rather weak BPDE removal of 50% of the BPDE adducts initially formed binding anities, guanines on the transcribed strand at at that site. At most sites on the transcribed strand it or near these codon positions (158, 244 and 282) have takes only *5 h to remove 50% of the BPDE adducts, relatively high BPDE binding anities. The cause for except at codon 252 where *10 h is required. In such dierences in BPDE binding between transcribed contrast, it generally takes 10 ± 20 h, to remove 50% of and nontranscribed strands is unknown. It is possible the BPDE adducts from sites on the nontranscribed that at such sequences the transcribed strand may face strand, except at codon 181, where it takes only 5 h. out of the nucleosome, while the nontranscribed strand may face into the nucleosome, therefore, BPDE binding in the latter, but not in the former, will be Discussion interfered with by the nuclear proteins. Considerable BPDE binding, either in one or both strands, was also Two factors which may contribute to site- and observed at codons 170, 242, 283, and 290. While sequence-dependent mutations are: the frequency of signi®cant numbers of mutations have been found at DNA damage at a particular site, and the eciency of codons 242 and 283 in lung cancer, very few mutations repair of damage formed at that site. Using UvrABC- Slow repair of DNA adducts in the human p53 gene MF Denissenko et al 1243 upper lower

00004824 hours repair 00004824 hours repair CG 00222 2 2 BPDE (µM) CG 00222 2 2 BPDE (µM) T A G –+–++++ UvrABC TA –+–++++ UvrABC

154

G* 170 T 157 C

158

156

175

181

12345 67889910 123456 7 Figure 1 Distribution and repair of BPDE-DNA adducts along sequences of exon 5 of the p53 gene in human ®broblasts. Cultured cells were treated with 2 mM BPDE for 30 min and incubated for various time periods to allow repair. DNAs isolated from these cells were treated with UvrABC and then subjected to LMPCR to determine the relative frequencies of adducts formed at various sites, on either (a) the nontranscribed (upper) strand or (b) transcribed (lower) strand of exon 5. Maxam and Gilbert sequencing ladders (Maxam & Gilbert, 1980) are indicated as G, GA and TC. The brackets indicate positions of selected p53 codons. The guanine at the lung cancer mutational hotspot at codon 157 (asterisk) is a site of slow repair

LMPCR methods, we have found that the chemically in this work show that BPDE indeed forms DNA activated cigarette smoke component BPDE forms adducts at these mutational hotspots (codons 157, 248 DNA adducts preferentially at sites along the and 273), on both the transcribed and nontranscribed nontranscribed strand of the p53 gene which had strands of the p53 gene. However, we found that repair been previously identi®ed as mutational hotspots in of BPDE adducts formed in the nontranscribed strand human lung cancer (Denissenko et al., 1996). Interest- at these mutational hotspots is two to four times ingly all of these preferential BPDE binding sites are slower than repair of adducts formed in the transcribed located at CpG sequences. Our further studies have strand. Both, preferential binding and slow repair of indicated that cytosine methylation at such CpG BPDE adducts formed in the nontranscribed strand at sequences is the major factor leading to this enhanced mutational hotspot sites in the p53 gene may be the BPDE-DNA binding (Denissenko et al., 1997). We major reason leading to the strand-bias of G to T expected that BPDE should also preferentially form transversion in lung cancer (Greenblatt et al., 1994). adducts in the transcribed strand at these mutational Transcribed strand biased repair of cyclobutane hotspot sites in the p53 gene since both DNA strands pyrimidine dimers and bulky chemicals induced DNA have the same methylation pattern at such sites adducts has been observed in human cells (Hanawalt, (Tornaletti and Pfeifer, 1995). The results we present 1994; Hanawalt et al., 1994; Bohr, 1991). While it has Slow repair of DNA adducts in the human p53 gene MF Denissenko et al 1244 upper lower

0000 4824 hours repair 000 04824hours repair CG0 02 2 222BPDE (µM) CG0 022222BPDE (µM) T A --+++++UvrABC T A --+++++UvrABC

248 242

244 244

241

C G* 248 G*

123456789 123456789 Figure 2 Distribution and repair of BPDE-DNA adducts along sequences of exon 7 of the p53 gene in human ®broblasts. The relative frequencies of DNA adducts formed at various sites, on either (a) the nontranscribed (upper) strand or (b) transcribed (lower) strand, were analysed by the same methods described in Figure 1. Maxam and Gilbert sequencing ladders (Maxam and Gilbert, 1980) are indicated as GA and TC. The brackets indicate positions of selected p53 codons. The two guanines at the lung cancer mutational hotspot codon 248 (asterisks) are sites of slow repair. Repair of damage on the transcribed (lower) strand at this codon is considerably faster

been reported that BPDE-adducts are more eciently carcinogens used (which might dierentially aect repaired in the transcribed strand than in the transcription initiation), or regional dierences in the nontranscribed strand of human HPRT gene (Chen relative eectiveness of global repair versus transcrip- et al., 1992), these adducts are repaired with equal tion coupled repair mechanisms, may also play eciency in both strands of DHFR and APRT genes in important roles in determining strand bias of repair. cultured Chinese hamster ovary cells (Tang et al., In general, the eects of transcription on repair are 1994). BPDE binds not only to nucleic acids but also more pronounced at low levels of DNA damage to nucleophilic proteins; BPDE-DNA adducts have (Vrieling et al., 1989, 1991). Since much higher levels been shown to bind to transcription factors (Treiber et of DNA damage are typically employed for repair al., 1994; Butler et al., 1997). Such interactions may assays than for mutation assays, this may be the reason interfere with initiation of transcription, and the that the strand bias of mutation is much more initiation of transcription of individual genes might pronounced than the strand bias of repair. For be subject to dierent degrees of inhibition by the same example, 90% of G to T transversions in the p53 chemical. This might explain dierences in the extent gene occur in the nontranscribed strand (Greenblatt et of strand biased repair observed for individual genes. al., 1994), however, we observed only 2 ± 4-fold Other factors, such as the concentrations of the dierence in repair between the two strands. Slow repair of DNA adducts in the human p53 gene MF Denissenko et al 1245 upper lower

00004824 hours repair 00004824 hours repair CG0022222 BPDE (µM) CG0022222 BPDE (µM) T A --+ ++++ UvrABC T A --+++++UvrABC

290

C G* 273 T 276

283

282

273

123456789 123456789

Figure 3 Distribution and repair of BPDE-DNA adducts along sequences of exon 8 of the p53 gene in human ®broblasts. The relative frequencies of DNA adducts formed at various sites, on either (a) the nontranscribed (upper) strand or (b) transcribed (lower) strand, were analysed by the same methods described in Figure 1. Maxam and Gilbert sequencing ladders (Maxam and Gilbert, 1980) are indicated as GA and TC. The brackets indicate positions of selected p53 codons. The guanine at the lung cancer mutational hotspot at codon 273 is indicated by an asterisk

Our results show that BPDE adducts are removed either the transcribed or nontranscribed strand using two to four times faster from the transcribed strand the UvrABC-LMPCR method. However, when BPDE than from the nontranscribed strand of the p53 gene. adducts are assayed directly by UvrABC in BPDE This result is consistent with the hypothesis that the modi®ed puri®ed p53 DNA without using the LMPCR process of transcription may facilitate nucleotide technique, a high level of BPDE adducts is found at excision repair (Hanawalt, 1994; Hanawalt et al., codon 245 (data not shown). These results indicate that 1994). However, our results also demonstrate that our failure to detect BPDE adduct formation at codon repair eciency is DNA sequence-dependent, and that 245 using the UvrABC-LMPCR method is probably it is more variable in the nontranscribed strand than in not due to inability of UvrABC nuclease to cut adducts the transcribed strand of the p53 gene. Transcription- formed at this sequence, but rather, may be a coupled nucleotide excision repair may have equal consequence of either the DNA polymerase being accessibility to all BPDE-DNA adducts in the unable to fully extend the UvrABC incised DNA or transcribed strand owing to sequence-indendent block- this fully extended DNA being refractory to ligation. age of RNA polymerase at these adducts. Although BPDE alkylates the guanine residue at Although codon 245 of the p53 gene is a mutational codon 156(-CGC- as strongly as the one at codon 157 hotspot in human cancer (including lung cancer), no (-GTC-) in the nontranscribed strand, and the BPDE strong BPDE adduct formation is found at this site on adducts formed at both these sequences are repaired Slow repair of DNA adducts in the human p53 gene MF Denissenko et al 1246 skin cancer mutational hotspots in the p53 gene (Tornaletti and Pfeifer, 1994). The current results provide another example showing that both DNA damage and repair rates are important determinants of sequence-dependent mutation rates in human genes.

Materials and methods

Cell culture and BPDE treatment Normal human ®broblasts were grown under standard conditions (Denissenko et al., 1996, 1997). Cells growing at con¯uency were treated with BPDE (2 mM)for30min; after various incubation times cells were harvested and DNA isolated. Methods used for BPDE treatment and DNA isolation were the same as previously described (Denissenko et al., 1996, 1997). No DNA replication was observed under these conditions (Venkatachalam et al., 1995). It has been well established that the guanine adducts are the major adducts formed in cultured cells or DNA treated with racemic mixtures of BPDE, and 80 to 90% of the guanine adducts are the (+) optical isomers (MacLeod & Tang, 1985; MacLeod et al., 1988; Stevens et al., 1985).

Treatment with UvrABC Uvr proteins were puri®ed as described (Tang, 1996). Puri®ed DNA was treated with an excess of UvrABC (a tenfold molar excess of protein over 104 nucleotides of DNA) as described (Tang, 1996). Under the reaction conditions used, cleavage at BPDE-DNA adducts by Figure 4 Quantitation of the rates of repair of BPDE adducts in UvrABC nucleases is quantitative. After UvrABC reac- the nontranscribed strand (upper) and transcribed strand (lower) tion, the Uvr proteins were removed by phenol extractions along exons 5, 7, and 8 of the p53 gene. Repair rates, determined as the time at which 50% of the BPDE signal was removed, were followed by diethyl ether extractions, and DNA was estimated for each position with a visible signal above back- ethanol precipitated and resuspended in TE solution ground from autoradiographs such as those shown in Figures 1 ± (1 mM Tris-HCl pH 8.0 and 0.1 mM EDTA). 3. Background values (from the 0 mM BPDE+UvrABC lanes) were substracted. A repair time course was established for each position, and the time at which 50% of the initial damage was Ligation-mediated PCR (LMPCR) and quantitations removed was then determined from this curve. The rates of repair The conditions and oligonucleotide primers used for at each site are represented by vertical columns. These data LMPCR of the human p53 gene are described elsewhere represent the average of two to three experiments for each exon (Tornaletti et al., 1993; Tornaletti & Pfeifer, 1994). After and DNA strand reaction the ampli®ed DNAs were separated by denaturing polyacrylamide gel electrophoresis, electroblotted to a nylon membrane, and then hybridized with 32P-labeled probes. The nylon membranes were exposed to a with an equally slow rate (Figure 1 and 4), codon 157 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). is a mutational hotspot in human lung cancer but Radioactivity was determined in all BPDE-speci®c bands codon 156 is not (Greenblatt et al., 1994; Harris, 1996). of the sequencing gel that could be resolved and showed a In fact, none of the -CGC- sites, including codon 175, quanti®able signal after substraction of background values. in the p53 gene are hotspots for mutation in lung cancer (Greenblatt et al., 1994; Harris, 1996). Possible explanations for these results are: (1) BPDE adducts Abbreviations formed at -CGC- sequence may allow only a very low BP, benzo[a]pyrene; BPDE, (+) anti-7b,8a-dihydroxy-9a, probability of translesion DNA synthesis and/or a very 10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; LMPCR, li- high ®delity of translesion DNA synthesis; (2) the gation-mediated polymerase chain reaction; EDTA, ethy- amino acid change resulting from the G to T lenediaminetetraacetic acid; HPRT, hypoxanthine-guanine transversion occurring at codon 156 may have little phosphoribosyltransferase; DHFR, dihydrofolate reduc- eect on p53 function; in fact, only one G to T tase; APRT, adenine phosphoribosyltransferase. transversion is found at this codon in all cancers combined (Greenblatt et al., 1994). Further studies are needed to test these possibilities. In previous work, repair of BPDE adducts was Acknowledgements We are grateful for Mr Yi Zheng's assistance in preparing found to be sequence-dependent in the human HPRT Uvr proteins, for S Bates' assistance with cell cuture, and gene and slow repair of speci®c positions in the gene for critical reviews of this manuscript by Drs Gerald Adair was correlated with mutational hotspots (Wei et al., and Yen-Len Tang. This study was supported by National 1995). Similarly, it has been shown that U.V.-induced Institutes of Health Grants ES03124, ES08389, CA65652, cyclobutane pyrimidine dimers are repaired slowly at and P01CA69449. Slow repair of DNA adducts in the human p53 gene MF Denissenko et al 1247 References

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