Nucleic Acids Research, 2000, Vol. 28, No. 23 4717-4724
© 2000 Oxford University Press
Error-prone lesion bypass by human DNA polymerase 
Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA, 1Chemistry Department, New York University, New York, NY 10003, USA and 2Department of Chemistry, Washington University, St Louis, MO 63130, USA
Received August 8, 2000; Revised and Accepted October 17, 2000.
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ABSTRACT |
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DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase
(Pol), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Pol, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Pol efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Pol effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5' to the abasic site is a T. Human Pol partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N2-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N2-dG lesion in mammalian cells. These results show that human Pol is capable of error-prone translesion DNA syntheses in vitro and suggest that Pol may bypass certain lesions with a mutagenic consequence in humans. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lesion bypass is an important cellular response to unrepaired DNA damage during replication. Two modes of lesion bypass are known, error-free bypass and error-prone bypass. Error-free lesion bypass results in the preferential incorporation of the correct nucleotide opposite the damage, whereas error-prone lesion bypass leads to the preferential incorporation of an incorrect nucleotide opposite the damage. Consequently, error-free lesion bypass is a mutation-avoiding mechanism, whereas error-prone lesion bypass is a mutation-generating mechanism. In eukaryotes, an important error-prone lesion bypass mechanism, also known as the damage-induced mutagenesis pathway, has been discovered originally in the yeast Saccharomyces cerevisiae (1–5). Later, this mechanism was also found in humans (5–9). In this mutagenesis pathway, DNA polymerase
(Pol) (the REV3–REV7 protein complex) and the REV1 dCMP transferase are involved in the translesion DNA synthesis step (7,10,11).
More recently, it has been demonstrated that Pol is involved in error-free lesion bypass of a TT dimer (12–14), which apparently operates independently of the Pol mutagenesis pathway (15). Pol is encoded by the RAD30 gene in the yeast S.cerevisiae and the Xeroderma pigmentosum variant (XPV) gene in humans (12,13). Pol plays an important role in response to UV radiation in humans, since a defect in this gene will lead to the hereditary XPV disease. XPV patients exhibit sensitivity to the sunlight and a predisposition to skin cancer (16). The molecular pathology of XPV could be attributed to the function of Pol in error-free lesion bypass of TT dimers and perhaps other cyclobutane pyrimidine dimers (CPD) as well. One molecular explanation for XP disease is as follows. During replication, some unrepaired CPDs would be bypassed by Pol without mutations. In the absence of Pol, CPDs that are normally bypassed by Pol would accumulate during replication. Some of those CPDs may lead to cell death, resulting in UV sensitivity; while other CPDs may be bypassed by the Pol mutagenesis pathway, resulting in an elevated frequency of UV mutations. XPV cells show a high proportion of mutations at cytosine-containing photoproducts and a strand bias for mutation specificity (17). These observations remain unexplained.
Most recently, translesion syntheses opposite a cisplatin adduct and an acetylaminofluorene-adducted guanine (AAF-G) have been observed with purified human Pol in vitro (18,19) (our unpublished results). These observations, together with our studies on yeast Pol (20), raised the possibility that human Pol may be capable of bypassing other DNA lesions. Furthermore, it is not very clear whether human Pol is specifically an error-free lesion bypass polymerase. To address these questions, we have examined the response of purified human Pol to several kinds of DNA damage in vitro. In this report, we show that human Pol is capable of error-prone bypass opposite an 8-oxoguanine lesion, an apurinic/apyrimidinic (AP) site and a (+)-trans-anti-benzo[a]pyrene-N2-dG bulky adduct. Our results suggest that human Pol can bypass different types of DNA lesions during replication, but certain lesions are bypassed with mutagenic consequences.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials
A mouse monoclonal antibody against the His6 tag was obtained from Qiagen (Valencia, CA). Alkaline phosphatase conjugated anti-mouse IgG was obtained from Sigma Chemicals (St Louis, MO). Platinum High Fidelity Tag DNA polymerase was purchased from BRL (Bethesda, MD). The human T cell ZAP Express cDNA library was purchased form Stratagene (La Jolla, CA). The yeast rad30 deletion mutant strain BY4741rad30
(MATa his3 leu2 met15 ura3 rad30) was from Research Genetics (Huntsville, AL).
Damaged DNA templates
A 30mer DNA template containing a site-specific 8-oxoguanine was synthesized via automated DNA phosphoramidite methods by Operon (Alameda, CA). The sequence is 5'-GGATGGACTGCAGGATCCGGAGGCCGCGCG-3', where the position of the 8-oxoguanine is underlined. The 36mer templates containing a site-specific tetrahydrofuran (AP site analog) were also synthesized by Operon. The sequences are 5'-GAAGGGATCCTTAAGACTXTAACCGGTCTTCGCGCG-3', 5'-GAAGGGATCCTTAAGACAXTAACCGGTCTTCGCGCG-3', 5'-GAAGGGATCCTTAAGACGXTAACCGGTCTTCGCGCG-3', 5'-GAAGGGATCCTTAAGACCXTAACCGGTCTTCGCGCG-3', where X designates the AP site. A 49mer DNA template containing a site-specific cis-syn TT dimer or a TT (6-4) photoproduct was prepared as previously described (21). Its sequence is 5'-AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3', where the modified TT is underlined. A 33mer DNA template, 5'-CTCGATCGCTAACGCTACCATCCGAATTCGCCC-3', was reacted with (+)-7R,8S-dihydrodiol-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-trans-anti-BPDE] to generate the 10S (+)-trans-anti-BPDE-N2-dG adduct site at the underlined G and purified as previously described (22–24).
Overexpression plasmid of the human XPV gene
The human XPV cDNA was obtained by polymerase chain reaction (PCR) amplification from the human T cell ZAP Express cDNA library using Platinum High Fidelity Tag DNA polymerase and two primers, 5'-CGGGATCCATGGCTACTGGACAGGATCGAG-3' and 5'-ACGCGTCGACCATTGTACCCGGCCGAG-3'. The resulting 2.6 kb PCR product was then cloned into the BamHI and SalI sites of the vector pECUh6, yielding pECUh6–XPV. The human XPV gene was verified by DNA sequencing. This expression construct contained the 2 µm origin for multicopy plasmid replication, the URA3 gene for plasmid selection, the CUP1 promoter for inducible XPV gene expression and six His codons preceding the ATG initiator codon of the XPV gene.
Purification of human DNA Pol
Yeast rad30 deletion mutant cells containing pECUh6–XPV were grown at 30°C for 2 days in minimum medium containing 2% dextrose. After 10-fold dilution in 16 l of YPD (2% Bacto-peptone, 1% yeast extract, 2% dextrose) medium, cells were grown for 6 h at 30°C. Expression of human Pol was induced by adding CuSO4 to 0.3 mM and grown for another 3 h. Cells were collected by centrifugation and washed in water. After resuspending in an extraction buffer containing 50 mM Tris–HCl pH 7.5, 1 M KCl, 10% sucrose, 20% glycerol, 5 mM ß-mercaptoethanol and protease inhibitors (25), cells were homogenized by zirconium beads in a bead-beater for 15 pulses of 30 s each on ice. The clarified extract (
130 ml) was loaded onto a HiTrap chelating column charged with NiSO4 (Amersham Pharmacia, 10 ml), followed by washing the column sequentially with 100 ml of Ni buffer A (20 mM phosphate buffer pH 7.4, 1 M KCl, 10% glycerol, 5 mM ß-mercaptoethanol and protease inhibitors) containing 10 mM imidazole and 100 ml of Ni buffer A containing 35 mM imidazole. Bound proteins were eluted with a linear gradient of 35–108 mM imidazole. The His6-tagged human Pol was identified by western blot using a mouse monoclonal antibody specific to the His6 tag. The pooled nickel column sample was concentrated by PEG 10 000 and desalted through five 5-ml Sephadex G-25 columns in FPLC buffer A (50 mM Tris–HCl pH 7.5, 1 mM EDTA, 10% glycerol and 5 mM ß-mercaptoethanol) containing 100 mM KCl. The resulting sample (40 ml) was loaded onto a Mono S HR5/5 column (Amersham Pharmacia) and eluted with a 30-ml linear gradient of 100–400 mM KCl in FPLC buffer A. Pol was eluted at 250 mM KCl. Fractions containing Pol were pooled and concentrated by PEG 10 000. Then, the sample was loaded onto an FPLC Superdex 200 gel filtration column equilibrated with FPLC buffer A containing 300 mM KCl and the column was developed in the same buffer.
DNA lesion bypass assays
Lesion bypass assays were performed in standard DNA polymerase reactions using various damaged DNA templates as indicated in the text. The standard DNA polymerase reaction (10 µl) contained 25 mM potassium phosphate pH 7.0, 5 mM MgCl2, 5 mM dithiothreitol, 100 µg/ml bovine serum albumin, 10% glycerol, 50 µM dNTPs (dATP, dCTP, dTTP and dGTP individually or together as indicated), 50 fmol of a DNA substrate containing a 32P-labeled primer and purified DNA Pol. After incubation at 30°C for 10 min, reactions were terminated with 7 µl of a stop solution (20 mM EDTA, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol). The reaction products were resolved on a 20% polyacrylamide gel containing 8 M urea and visualized by autoradiography. Primer extension was quantitated by scanning densitometry of the autoradiogram using the SigmaGel software (Sigma) for analysis.
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RESULTS |
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Purification of human DNA Pol
To facilitate protein purification and detection, we tagged human Pol
with six histidine residues at its N-terminus. The tagged protein was expressed in yeast cells of the rad30 deletion mutant strain to avoid potential contamination by the yeast Pol. Human Pol was then purified to near homogeneity (Fig. 1A). The identity of the tagged human Pol was confirmed by western blot analysis using a mouse monoclonal antibody specific to the His6 tag (Fig. 1B). The purified human Pol migrated as a 77 kDa protein on a 10% SDS–polyacrylamide gel (Fig. 1A), consistent with its calculated molecular weight of 78 kDa. Using a 49mer DNA template containing a site-specific cis-syn TT dimer or a TT (6-4) photoproduct (Fig. 2A), we performed lesion bypass assays with the purified human Pol. A 32P-labeled 15mer primer was annealed right before the UV lesion (Fig. 2A). As expected, the purified human Pol efficiently bypassed the TT dimer (Fig. 2B), but was unable to bypass the TT (6-4) photoproduct (Fig. 2C, lane 2). After incorporating one nucleotide opposite the 3' T of the TT (6-4) photoproduct, DNA synthesis by human Pol was completely stopped (Fig. 2C, lane 2). To reveal the identity of this incorporated nucleotide, we performed DNA synthesis assays with only one deoxyribonucleoside triphosphate: dATP, dCTP, dTTP or dGTP individually. As shown in Figure 2C (lanes 3–6), human Pol predominantly incorporated a G opposite the 3' T of the TT (6-4) photoproduct. Less frequently, an A was also incorporated (Fig. 2C, lane 3). These results show that our purified human Pol is a full-length protein and an active enzyme.
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Error-prone bypass of template 8-oxoguanine by human Pol
8-Oxoguanine is a major form of oxidative damage in DNA. To examine whether human Pol
can bypass this lesion, we synthesized a 30mer DNA template containing a site-specific 8-oxoguanine residue (Fig. 3). A 32P-labeled 17mer primer was annealed to the template, right before the 8-oxoguanine residue (Fig. 3A). As shown in Figure 3A (lane 6), human Pol efficiently bypassed the template 8-oxoguanine. To identify the base incorporated opposite 8-oxoguanine, we performed DNA synthesis assays with only one deoxyribonucleoside triphosphate: dATP, dCTP, dGTP or dTTP individually. As shown in Figure 3A (lanes 7–10), human Pol extended 79% of the primers using dATP, 75% of the primers using dCTP and 32% of the primers using dGTP opposite the template 8-oxoguanine. In comparison, human Pol predominantly incorporated the correct C opposite the undamaged template G (Fig. 3A, lanes 1–5). We consistently observed that copying the last template base by human Pol from undamaged DNA was not very efficient, often resulting in two bands visible after separation on a denaturing polyacrylamide gel (e.g. Fig. 3A, lane 1).
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Misincorporation of A opposite template 8-oxoguanine would lead to G
T transversion. To examine whether the misincorporated A could be extended by human Pol, we separately annealed two 32P-labeled 18mer primers to the 8-oxoguanine DNA template, forming an 8-oxoguanine-A mismatch and an 8-oxoguanine-C base pair at the primer 3'-end, respectively (Fig. 3B). As shown in Figure 3B (lanes 1 and 6), both primers were efficiently extended by human Pol. In both cases, the correct nucleotide T was predominantly incorporated opposite the undamaged template base A 5' to the lesion by human Pol (Fig. 3B, lanes 4 and 9). Human Pol extended 52 and 54% of the 8-oxoguanine-A and the 8-oxoguanine-C base pairs, respectively, using dTTP (Fig. 3B, lanes 4 and 9).
To obtain a more quantitative comparison between A and C incorporations opposite the template 8-oxoguanine and subsequent extensions to the 5' undamaged template base, we performed steady-state kinetic analyses using a previously described method (26). As shown in Table 1, the template 8-oxoguanine slowed human Pol 5.3-fold with respect to C incorporation, as indicated by the finc (0.19). However, human Pol incorporated both C and A opposite the 8-oxoguanine with essentially the same efficiency (Table 1). Consequently, A misincorporation by human Pol increased 421-fold (3.8 x 10–4/1.6 x 10–1) from opposite undamaged template G to 8-oxoguanine (Table 1). Further extension of the misincorporated A from opposite the lesion to the next template base was only 1.7-fold (1/5.8 x 10–1) slower than extension from the correct 8-oxoguanine-C base pair (Table 1). Therefore, we conclude that, upon encountering 8-oxoguanine in DNA, human Pol will perform efficient error-prone lesion bypass, leading to frequent GT transversions.
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Mutagenic translesion synthesis opposite a template AP site by human Pol
AP sites are significant DNA lesions, which can arise in the genome spontaneously or can be induced by many environmental agents. Using purified human Pol
, we examined the response of this polymerase to an AP site in DNA. A 14mer primer was labeled at its 5'-end with 32P and annealed to a DNA template (template AP-T). The 3'-end of the primer was annealed three nucleotides before the template AP site (Fig. 4). As shown in Figure 4 (lane 3), purified human Pol was able to bypass this AP site. Human Pol efficiently incorporated one nucleotide opposite the template AP site, but its further extension was inhibited by the lesion (Fig. 4, lane 3), as evidenced by the accumulation of the 18mer synthesis products. In the absence of the AP site, the 18mer synthesis product was not accumulated by human Pol (Fig. 4, lane 2). By increasing the amount of human Pol from 21 to 103 fmol in the reaction, the majority of the 14mer primers were extended to the end of the template within 10 min at 30°C, bypassing the template AP site (data not shown).
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Bypass of AP sites by human Pol
was strongly influenced by the sequence context 5' to the AP site (27,28). To examine whether AP site bypass by human Pol is also influenced by sequence context, we synthesized three more DNA templates that differed from the above AP site template (template AP-T) by one nucleotide 5' to the AP site (Fig. 5A). A 17mer primer was labeled at its 5'-end with 32P and annealed to the four AP site templates, right before the template AP site (Fig. 5A). Then, the primed DNA templates were incubated with human Pol for lesion bypass assays. As shown in Figure 5B, the efficiency of AP site bypass by human Pol was similar among the four templates. Again, primer extension was inhibited after incorporating one nucleotide opposite the AP site (18mer DNA fragment) (Fig. 5B, lanes 2–5).
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When the template base 5' to the AP site is a T (template AP-T), the majority of the bypassed products appeared to be one nucleotide shorter (Fig. 5B, lane 4). This result raised the possibility that deletions might be involved during AP site bypass by human Pol
in the AP-T template. To examine this possibility, we performed lesion bypass assays followed by digestion with the restriction endonuclease AflII. For normal primer extension and after AflII digestion of the extended products, a 32P-labeled 22mer band was expected to arise (Fig. 5A). Indeed, using the undamaged template (template 18T), DNA synthesis products of human Pol (Fig. 6, lane 1) were cleaved by AflII to a 22-nt fragment (Fig. 6, lane 2). However, using the AP site template AP-T, lesion bypass products of human Pol (Fig. 6, lane 7) were cleaved by AflII to three bands, 22mer, 21mer and 20mer fragments with relative intensities of 6.7:7.7:1, respectively (Fig. 6, lane 8). Thus, 44, 50 and 6% of the bypass products were derived from bypass without deletion, –1 deletion and –2 deletion mechanisms, respectively. Using AP site templates AP-A, AP-C and AP-G containing a template A, C or G, respectively, 5' to the AP site (Fig. 5A), we performed similar AflII cleavage experiments following lesion bypass by human Pol. As shown in Figure 6 (lanes 4, 6 and 10), the major cleavage product was the 22mer DNA fragment, indicating bypass without deletion.
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To identify the nucleotide incorporated by human Pol
opposite the template AP site, we performed lesion bypass assays with only one deoxyribonucleoside triphosphate. As shown in Figure 7, nucleotide incorporation opposite the AP site by human Pol followed the order from most frequent to least frequent: A>G>C>T. Taken together, these results show that human Pol mainly incorporates A opposite a template AP site, whose further extension is the rate-limiting step. When the template base 5' to the AP site is a T, 50% of the AP site bypass is mediated by a –1 deletion mechanism.
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Error-prone translesion synthesis opposite a (+)-trans-anti-benzo[a]pyrene-N2-dG adduct by human Pol
Like a TT dimer, (+)-trans-anti-benzo[a]pyrene-N2-dG is a bulky lesion in DNA. This lesion represents the major DNA damage caused by the reaction of racemic anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a potent ultimate carcinogen of benzo[a]pyrene, with DNA (29,30). Using purified human Pol
, we examined the response of this polymerase to a template (+)-trans-anti-BPDE-N2-dG bulky lesion. A 19mer primer was labeled with 32P at its 5'-end and annealed right before the template (+)-trans-anti-BPDE-N2-dG lesion (Fig. 8). As shown in Figure 8 (lane 1), human Pol effectively incorporated one nucleotide opposite the lesion and extended one more nucleotide downstream, generating a 21mer DNA fragment. However, further DNA synthesis was inhibited by the lesion. Nevertheless, some of the stalled 21mer DNA fragments were further extended by human Pol to near the end of the DNA template (Fig. 8, lane 1). To identify the nucleotide incorporated opposite the lesion, we performed lesion bypass assays with only one of the four deoxyribonucleoside triphosphates at a time. As shown in Figure 8 (lanes 2–5), human Pol predominantly incorporated an A opposite the template (+)-trans-anti-BPDE-N2-dG. Less frequently, a T was incorporated opposite this lesion. Least frequently, a C or a G was also incorporated opposite this lesion. These results demonstrate that human Pol is capable of error-prone translesion synthesis opposite a template (+)-trans-anti-BPDE-N2-dG.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pol
was originally identified as an error-free lesion bypass polymerase in response to UV radiation (12–14). This activity of human Pol is critical in preventing the XPV disease (13,31). However, our recent biochemical studies on the yeast Pol suggest that this polymerase may also be involved in error-prone translesion synthesis (20). In this study, we show that human Pol is indeed able to bypass an 8-oxoguanine, an AP site and a (+)-trans-anti-benzo[a]pyrene-N2-dG bulky adduct in an error-prone manner in vitro. Hence, depending on the specific DNA lesion, human Pol is capable of both error-free and error-prone translesion syntheses, at least in vitro.
Error-free AA incorporations opposite a TT dimer by human Pol can be interpreted by two mechanisms. One is indiscriminating A incorporation opposite a damaged template. The other is that the TT dimer is recognized by Pol as a coding TT sequence. We found that human Pol predominantly incorporates a G opposite the 3' T of a TT (6-4) photoproduct. This result supports the second mechanism of lesion bypass by Pol and is inconsistent with the first interpretation. An intriguing observation is that a site-specific TT (6-4) photoproduct induced primarily 3' TC substitution mutations in COS cells, resulting from G incorporation opposite the 3' T of the lesion (32). Since human Pol did not bypass the 5' T of the TT (6-4) photoproduct at the enzyme concentrations used, it is not clear at present whether G incorporation by human Pol opposite the 3' T of the lesion is physiologically significant. However, it is possible that, following G incorporation, the 5' T of the lesion may be bypassed by human Pol. Supporting this model, cooperation between yeast Pol and yeast Pol to bypass a template AP site has been observed in vitro (20). Furthermore, when a primer 3'-end is a G and annealed opposite the 3' T of the TT (6-4) photoproduct, this primer is effectively extended to the end of the template by purified yeast Pol (D.Guo, and Z.Wang, unpublished results).
Significant AP site bypass was observed at a low polymerase to DNA ratio: 21 fmol Pol versus 50 fmol DNA (Fig. 4). Thus, it is likely that human Pol plays a role in error-prone bypass of AP sites in vivo. The consequence of AP site bypass by human Pol is mainly A incorporation opposite the lesion, although less frequently, G is also incorporated. When the template base 5' to the AP site is a T, a –1 deletion was observed among 50% of the bypass products. This –1 deletion bypass probably results from re-alignment of the incorporated A with the next template T. This property of human Pol is reminiscent of the AP site bypass by human Pol (27,28). However, in contrast to human Pol (27), the efficiency of AP site bypass by human Pol is not significantly affected by the sequence context 5' to the AP site. AP site bypass by human Pol was also observed by Masutani et al. (19). However, the –1 deletion bypass and the sequence context effect on AP site bypass by human Pol were not examined in the study of Masutani et al. (19).
In Escherichia coli, translesion synthesis of an AP site results in preferential incorporation of an A opposite the lesion, leading to the ‘A rule’ hypothesis (33). In mammals, however, it seems that A incorporation opposite an AP site is not strongly biased. Similar incorporations of A, C and T opposite an AP site were observed (34–37). In one study, preferential A incorporation opposite an AP site was noticed (38). Yet in another study, preferential G incorporation was detected opposite an AP site (39). Relative to A incorporation, G is also significantly incorporated by purified human Pol opposite an AP site, especially when the template base 5' to the lesion is a pyrimidine. Thus, Pol may have contributed in part to the previously reported A and G incorporations opposite a template AP site in mammalian cells.
The (+)-trans-anti-BPDE-N2-dG bulky adduct is highly mutagenic in COS cells (40,41). The mutagenic potential of (+)-trans-anti-BPDE-N2-dG lesions in different base sequence contexts has been investigated using site-specific mutagenesis methods in a variety of prokaryotic and eukaryotic cellular systems (40–45). In some or all of these studies, bypass polymerases may have played a role in the mutagenic bypass of this lesion. In this study we found that human Pol can insert incorrect bases opposite the (+)-trans-anti-BPDE-N2-dG lesion. Human Pol predominantly incorporates an A, less frequently a T and even less frequently a G or C opposite the lesion (Fig. 8). Therefore, if these single nucleotide insertion experiments are reflected in full-length primer extension, then these results predict that Pol causes predominantly GT transversions, less frequent GA transitions and even less frequent GC transversions. Similar mutagenic specificities were observed in COS cells (40,41). This correlation between in vitro and in vivo results suggest a role for Pol in mutagenic bypass of the (+)-trans-anti-BPDE-N2-dG lesion in mammalian cells. An earlier study has attempted to examine the cytotoxicity and mutagenesis induced by BPDE in cultured XPV cells and concluded that neither was affected without functional XPV gene (46). In light of our new biochemical information on the response of human Pol to the (+)-trans-anti-BPDE-N2-dG adduct in vitro, more rigorously controlled in vivo studies are needed to definitively answer whether Pol is important for in vivo bypass and mutagenesis of benzo[a]pyrene adducts.
Comparing the yeast and the human Pol, some differences were noticed. While yeast Pol predominantly incorporates C opposite 8-oxoguanine (20), human Pol inserts C and A with similar efficiencies. Whereas yeast Pol predominantly incorporates G opposite the AP site (20), human Pol prefers A. Nevertheless, both yeast and human Pol were capable of translesion synthesis opposite multiple different DNA lesions. As suggested by two earlier reports, the ability of Pol to bypass DNA lesions may have derived from its relaxed requirement for correct base pairing geometry at the polymerase active site (47,48). As a result of such a specialized function in lesion bypass, Pol synthesizes DNA from undamaged templates with extraordinarily low fidelity (47,48).
Our results show that human Pol is capable of error-prone translesion DNA syntheses in vitro. These results raised the possibility that human Pol may be involved in mutagenesis induced by certain DNA lesions such as AP sites and (+)-trans-anti-BPDE-N2-dG adducts in vivo.
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ACKNOWLEDGEMENTS |
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This work was supported by a THRI grant from the Tobacco and Health Research Institute of the University of Kentucky (Z.W.), a New Investigator Award in Toxicology from Burroughs Wellcome Fund (Z.W.), a NIH grant CA40463 (J.-S.T.) and a NIH grant CA20851 (N.E.G.).
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 859 323 5784; Fax: +1 859 323 1059; Email: zwang{at}pop.uky.edu
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REFERENCES |
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