Nucleic Acids Research, 2000, Vol. 28, No. 21 4138-4146
© 2000 Oxford University Press
Error-free and error-prone lesion bypass by human DNA polymerase 
in vitro
Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA, 1Department of Chemistry, Washington University, St Louis, MO 63130, USA and 2Chemistry Department, New York University, New York, NY 10003, USA
Received July 21, 2000; Revised and Accepted September 13, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Pol
encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Pol is a novel lesion bypass polymerase in vitro. Purified human Pol efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Pol most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Pol was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Pol effectively bypassed a template (–)-trans-anti-benzo[a]pyrene-N2-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Pol was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Pol plays an important role in both error-free and error-prone lesion bypass 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 and error-prone bypass. The former mechanism does not introduce mutations opposite the lesion, whereas the latter mechanism is frequently accompanied by mutations. Therefore, error-prone lesion bypass constitutes a major mechanism of DNA damage-induced mutagenesis in cells. In Escherichia coli, damage-induced mutagenesis is mainly mediated by the UmuCD pathway under the control of the SOS response (1,2). DNA polymerase (Pol) V composed of the UmuCD'2 protein complex is responsible for the translesion DNA synthesis step (3,4). This mechanism largely results in targeted mutations opposite the DNA lesion. Under genotoxic stress conditions, the SOS system additionally turns on the dinB gene that codes for DNA polymerase IV (5). This polymerase is involved in untargeted mutagenesis in E.coli, i.e. mutations that arise in undamaged regions of DNA (6).
In eukaryotes, a damage-induced mutagenesis pathway has been discovered, originally in the yeast Saccharomyces cerevisiae (7,8) and later in humans (9–13). In this mutagenesis pathway, DNA Pol
(the REV3–REV7 protein complex) and the REV1 dCMP transferase are involved in the translesion DNA synthesis step (11,14,15). More recently, it has been demonstrated that Pol
is also involved in translesion synthesis opposite several DNA lesions, notably TT dimer (16–18). However, genetic analysis in yeast has indicated that Pol and the Rev3 mutagenesis pathway are two independent mechanisms of lesion bypass in response to UV radiation (19). During bypass of UV lesions, the Pol mechanism is error-free, whereas the REV3 pathway is error-prone (16,17,19,20). This feature further distinguishes these two lesion bypass mechanisms. In response to some other DNA lesions, Pol can be error-prone as well (18,21,22). In yeast, Pol is encoded by the RAD30 gene (19,23). It is now clear that the E.coli UmuC, the E.coli DinB, the yeast Rev1 and the yeast Rad30 constitute a group of related proteins called the UmuC superfamily. Human homologs of DinB (24,25), Rev1 (11,12) and Rad30 (26–28) have recently been isolated. A direct role in lesion bypass has been demonstrated in vitro for UmuC (3,4), Rev1 (11,14) and Rad30 (16–18,27). Most recently, the human DINB1 protein was shown to be a DNA polymerase, variously designated as Pol (29) and Pol
(30). It is noted that the eighth human DNA polymerase that is different from DINB1 has been previously reported and named Pol (31). To avoid confusion, we will refer to the human DINB1 protein as Pol in this report.
Lesion bypass is a ubiquitous biological process and directly affects human health. It affects the cytotoxicity and mutageneity of DNA damaging agents, including many chemical carcinogens and radiations. Mutations are building blocks for evolution, hereditary diseases and cancer. A recent example for the latter two biological effects is demonstrated by the human hereditary disease xeroderma pigmentosum variant (XPV). This disease is caused by mutations that inactivate the XPV gene coding for the lesion bypass protein Pol (26,27). Loss of error-free lesion bypass of pyrimidine dimers by Pol results in elevated mutagenesis following UV radiation (32), sensitivity to the sunlight and a predisposition to skin cancer (33). Hence, understanding lesion bypass is a key to the understanding of carcinogenesis.
In addition to the REV3 pathway and Pol, it is not known whether there is another lesion bypass mechanism in humans. Most recently, in vitro studies of apurinic/apyrimidinic (AP) site bypass by human Pol have yielded contradictory conclusions (29,30). To gain an insight into the function of Pol in human biology, we have studied this DNA polymerase in response to six different types of DNA lesions. In this report, we demonstrate that human Pol is a novel lesion bypass polymerase in vitro. Furthermore, we show that purified human Pol is capable of both error-free and error-prone lesion bypass, depending on the specific lesion.
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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 purchased from Qiagen (Valencia, CA). Alkaline phosphatase conjugated anti-mouse IgG was from Sigma (St Louis, MO). Pfu DNA polymerase was from Stratagene (La Jolla, CA). The E.coli Fpg protein was obtained from Trevigen (Gaithersburg, MA). The yeast rad30 deletion mutant strain BY4741rad30
(MATa his3 leu2 met15 ura3 rad30) was purchased from Research Genetics (Huntsville, AL). N-acetoxy-N-2-acetylaminofluorene (AAAF) was obtained from the Midwest Research Institute (Kansas City, MO). Human testis cDNA was purchased from Clontech Laboratories (Palo Alto, CA).
Damaged DNA templates
A 30mer DNA template containing a site-specific 8-oxoguanine was synthesized via automated DNA phosphoramidite methods by Operon (Alameda, CA). Its 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. Their sequences are 5'-GAAGGGATCCTTAAGACTXTAACCGGTCTTCGCGCG-3', 5'-GAAGGGATCCTTAAGACAXTAACCGGTCTTCGCGCG-3', 5'-GAAGGGATCCTTAAGACGXTAACCGGTCTTCGCGCG-3' and 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 (34). Its sequence is 5'-AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3', where the modified TT is underlined. A 33mer DNA template, 5'-CTCGATCGCTAACGCTACCATCCGAATTCGCCC-3', containing a site-specific (–)-trans-anti-benzo[a]pyrene [(–)-trans-anti-BPDE] adduct at the N2 position of the underlined G was prepared as previously described (35–37).
To prepare AAF-adducted DNA template, 2 nmol of the oligonucleotide 5'-CCTTCTTCTTCATACAAGCTTACTTCTTCC-3' was incubated with 200 nmol of N- AAAF at 37°C in the dark for 3 h in 100 µl of TE buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA) containing 20% ethanol. AAAF predominantly modified the unique G in the DNA. Free AAAF was removed by extracting the reaction mixture five times with water-saturated ether. Then, damaged and undamaged oligonucleotides were separated by electrophoresis on a 20% denaturing polyacrylamide gel. Acetylaminofluorene (AAF)-modified DNA migrated slower on the gel and was sliced out of the gel. The gel slices were soaked in 150 µl water at room temperature for 4 h. Finally, AAF-damaged DNA was recovered using GenElute DNA spin column (Sigma).
Human DINB1 gene and its overexpression
The vector pECUh6 was used to express human DINB1 gene in yeast cells. To construct pECUh6, the yeast CUP1 promoter sequence (269 bp) was amplified by polymerase chain reaction (PCR) from yeast genomic DNA using Pfu DNA polymerase and two primers: 5'-CGGAATTCTGTCAAGAGATTCTTTTGCTGGC-3' and 5'-CGGAATTCTCGAGTTTATGTGATGATTGATTGATTGATTGTAC-3'. The resulting PCR product was cloned into the EcoRI-digested pEGUh6 (38), replacing its GAL1/10 promoter sequence and yielding pECUh6. The yeast expression vector pECUh6 contains the 2 µm origin for multicopy plasmid replication, the URA3 gene for plasmid selection, the CUP1 promoter for inducible gene expression and six histidine codons for N-terminal protein tagging.
The human DINB1 cDNA was obtained by PCR amplification from human testis cDNAs using Pfu DNA polymerase and two primers: 5'-CGGGATCCATGGATAGCACAAAGGAGAAG-3' and 5'-CCCAAGCTTCACCTTCTTATCCCATTCCTAATTTC-3'. The resulting 2.9 kb PCR product was then cloned into the BamHI and HindIII sites of the vector pECUh6, yielding pECUh6–hDINB1. The entire sequence of the cloned human DINB1 cDNA was confirmed by DNA sequencing.
Purification of human DNA Pol
Yeast rad30 deletion mutant cells harboring pECUh6–hDINB1 were grown at 30°C for 2 days in minimum medium containing 2% dextrose. After 10-fold dilution in 16 l of YPD medium (2% bacto-peptone, 1% yeast extract, 2% dextrose), cells were grown for 6 h at 30°C. Expression of the human DINB1 gene was induced by adding CuSO4 to 0.3 mM and growth for another 3 h. Collected cells (
80 g) were homogenized by zirconium beads in a Bead-beater 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 (39). The clarified extract (130 ml) was loaded onto a HiTrap chelating column charged with NiSO4 (2 x 5 ml; Amersham Pharmacia Biotech, Piscataway, NJ), followed by washing the column sequentially with 100 ml of Ni buffer A (20 mM KH2PO4 pH 7.4, 1 M NaCl, 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 His-tagged human DINB1 protein was identified by western blots using a mouse monoclonal antibody specific to the His6 tag. The pooled sample was concentrated by PEG 10 000 and desalted through 5 x 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 an FPLC Mono S HR5/5 column (Amersham Pharmacia Biotech) and eluted with a 30 ml linear gradient of 100–400 mM KCl in FPLC buffer A. Human DINB1 protein was eluted at 250 mM KCl. The Mono S fractions were concentrated by PEG 10 000 and then loaded onto an FPLC Superdex 200 gel filtration column that had been equilibrated in FPLC buffer A containing 300 mM KCl. Human DINB1 protein was eluted at 100 kDa position.
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 human 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of human DNA Pol
The human DINB1 gene codes for a DNA polymerase (24,25,29,30), designated Pol
(29,40). To investigate its potential role in DNA lesion bypass, we purified human Pol. To facilitate its purification and detection, we tagged human Pol with six histidine residues at its N-terminus. The tagged human Pol 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). A faint band below human Pol was detected (Fig. 1A). It most likely is a C-terminal degradation product of human Pol, because this protein band was detected by the His6-specific antibody (Fig. 1B). The purified human Pol migrated at 81 kDa on a 10% SDS–polyacrylamide gel (Fig. 1A), faster than its calculated molecular weight of 99 kDa. Human Pol in freshly prepared cell extracts also migrated at a similar position on the 10% SDS–polyacrylamide gel (data not shown). Furthermore, the native molecular weight of the purified human Pol was determined to be 100 kDa by gel filtration chromatography on an FPLC Superdex 200 column. Hence, we conclude that our purified human Pol is a full-length protein.
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Using a 30mer DNA template and an annealed 17mer primer containing a 32P label at its 5'-end, we performed a DNA polymerase assay with the purified human Pol
. As shown in Figure 1C, the purified human Pol possesses a DNA polymerase activity. Primer extension by human Pol resulted in various sizes of DNA products that differed by one nucleotide in length (Fig. 1C, lane 2), indicating that it is a distributive polymerase. We consistently observed that human Pol-catalyzed DNA synthesis stops one or two nucleotides before the end of the template (Fig. 1C). This property of human Pol was also observed by Johnson et al. (30).
Translesion synthesis 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 DNA lesion, we synthesized a 30mer DNA template containing a site-specific 8-oxoguanine (Fig. 2). This DNA was then labeled with 32P at its 5'-end, annealed to its 30mer complementary strand and incubated with the E.coli 8-oxoguanine-DNA glycosylase (Fpg protein). Complete cleavage at the lesion site was observed (data not shown), thus confirming the identity and the position of the 8-oxoguanine residue in the synthesized DNA.
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To perform translesion synthesis assays, a 17mer primer was labeled with 32P at its 5'-end and subsequently annealed to the template, right before the 8-oxoguanine residue (Fig. 2). As shown in Figure 2 (lane 1), human Pol
efficiently bypassed the template 8-oxoguanine at a low polymerase concentration (4 fmol). The presence of 8-oxoguanine did not appear to significantly inhibit the activity of human Pol (Fig. 2, lane 1). This is in contrast to the significant blocking effect of a template 8-oxoguanine on the activity of purified human Pol
(our unpublished data). It is known that 8-oxoguanine is a miscoding DNA lesion, capable of base pairing with either a C or an A residue (41). To identify the base incorporated opposite 8-oxoguanine by human Pol, we performed DNA synthesis assays with only one deoxyribonucleoside triphosphate: dATP, dCTP, dGTP or dTTP individually. Human Pol extended 63% of the primers using dATP (Fig. 2, lane 2) and 53% of the primers using dCTP (Fig. 2, lane 3) opposite the template 8-oxoguanine. Primer extension was not detected with dTTP and dGTP (Fig. 2, lanes 4 and 5). These results suggest that human Pol predominantly incorporates an A residue opposite a template 8-oxoguanine. Hence, we conclude that when encountering 8-oxoguanine in DNA, human Pol will cause mutagenic translesion synthesis.
Mutagenic bypass 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. Since an AP site can derive from A, T, C or G, AP site bypass by a DNA polymerase will be error-prone. Thus, AP sites are potent mutagenic lesions in DNA. Using purified human Pol, we examined the response of this polymerase to an AP site in DNA. A 17mer primer was labeled at its 5'-end with 32P and annealed to a 36mer DNA template right before the template AP site. To additionally examine potential sequence context effect on AP site bypass, four templates were used that differed by one nucleotide 5' to the AP site (Fig. 3A). As shown in Figure 3B (lane 4), human Pol effectively bypassed the AP site in template AP–T that contained a T 5' to the AP site. When the template nucleotide 5' to the AP site was replaced with a C, A or G, the AP site was less efficiently bypassed by human Pol and one nucleotide insertion opposite the AP site was accumulated as the major product even at a high Pol concentration (20 ng, 200 fmol) (Fig. 3B, compare lanes 2, 3 and 5 with lane 4). Using the template AP–T containing a ‘running start’ primer annealed 3 nt before the template AP site, lesion bypass was also observed (Fig. 3C, lane 2). Human Pol activity was slightly inhibited right before and opposite the AP site in template AP–T during DNA synthesis, indicated by a small fraction of aborted synthesis as 17- and 18-nucleotide fragments (Fig. 3C, compare lane 1 with lane 2). These results show that human Pol is capable of translesion synthesis opposite a template AP site and that the bypass efficiency is greatly stimulated by a template T 5' to the AP site.
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To identify the nucleotide incorporated opposite the template AP site, we performed lesion bypass assays with only one deoxyribonucleoside triphosphate. As shown in Figure 4A–D (lane 2), A was most frequently inserted opposite the template AP site, which occurred at a low Pol
concentration (20 fmol) with the AP–T template, but required a high Pol concentration (200 fmol) with templates AP–G, AP–A and AP–C. The efficiency of C, T or G incorporation opposite the AP site was strongly influenced by the template base 5' to the AP site (Fig. 4A–D, lanes 3–5). With the template AP–A, significant C, T and G incorporations by human Pol were observed opposite the AP site, at levels only slightly lower than that of A incorporation (Fig. 4C, lanes 2–5).
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We consistently observed that DNA synthesis products are 1–2 nt shorter using template AP–T than those using the undamaged control template (template 18T) (Figs 3B, compare lane 1 with lane 4, 3C and 5A, compare lane 1 with lane 3). We suspected that these bypass products might contain deletions. To examine this possibility, we performed AP site bypass assays followed by digestion with the restriction endonuclease AflII. After AflII digestion, a 32P-labeled 22-nt band was expected with a normal primer extension (Fig. 3A). Indeed, using the undamaged template (template 18T), DNA synthesis products of human Pol
(Fig. 5A, lane 1) were cleaved by AflII to a 22-nt fragment (Fig. 5A, lane 2). Using the AP site template AP–T, lesion bypass products of human Pol (Fig. 5A, lane 3) were cleaved by AflII to a major 21-nt fragment and a minor 20-nt fragment (Fig. 5A, lane 4), indicating 1- and 2-nt deletions, respectively. Using AP site templates AP–A, AP–G and AP–C containing an A, G or C, respectively, 5' to the AP site (Fig. 3A), we performed similar AflII cleavage experiments following lesion bypass by human Pol. As shown in Figure 5B (lanes 2, 4 and 6), the major cleavage product was the 22-nt DNA fragment. A 21-nt DNA fragment (due to –1 deletion) was additionally observed as a minor AflII cleavage product with templates AP–A and AP–G (Fig. 5B, lanes 2 and 4). A 20-nt DNA fragment (due to –2 deletion) was observed as a minor AflII cleavage product with the template AP–C (Fig. 5B, lane 6). Consistent with these results, the major lesion bypass products with templates AP–A, AP–G and AP–C ranged from 33–35 nt, whereas those with the template AP–T ranged from 32–34 nt (Fig. 5A and B, compare lane 3 with lanes 1, 3 and 5, respectively).
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Taken together, these results show that human Pol
most frequently incorporates A opposite a template AP site. Efficient further extension requires T as the next template base and is mediated mainly by a one-nucleotide deletion mechanism. With an A, C or G template base 5' to the AP site, lesion bypass by human Pol is inefficient and is mediated mainly by a mechanism of A insertion followed by extension without deletion.
Error-prone lesion bypass opposite AAF-adducted guanine by human Pol
In contrast to AP sites, AAF adducts are bulky lesions in DNA. AAF-adducted guanines are the major AAF lesions in DNA, which block many DNA polymerases (18,42). To examine whether human Pol is able to bypass the bulky AAF-guanine lesion, we performed lesion bypass assays using a DNA template containing a site-specific AAF adduct. A 32P-labeled 13mer primer was annealed 4 nt before the AAF-adducted guanine (Fig. 6A). As shown in Figure 6A, human Pol was able to bypass the AAF adduct at a high polymerase concentration (200 fmol). However, a significant amount of DNA synthesis stopped after incorporating one nucleotide opposite the AAF-guanine (18-nt product) (Fig. 6A, lane 2). DNA synthesis stopping at this position was not observed with the undamaged control template (Fig. 6A, lane 1). Thus, the rate-limiting step of AAF bypass by human Pol is at the extension step after nucleotide incorporation opposite the lesion. Furthermore, the pattern of primer extension products is very similar at the end of both undamaged and AAF-damaged templates (Fig. 6A, compare lane 1 with lane 2). This result suggests that deletion is not a major mechanism for AAF bypass by human Pol at this sequence context.
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To identify the nucleotide incorporated opposite the AAF-guanine, we annealed a 32P-labeled 17mer primer right before the lesion and performed lesion bypass assays in the presence of only one deoxyribonucleoside triphosphate. As shown in Figure 6B (lane 2), effective bypass of the AAF-guanine was also observed when human Pol
was reduced to 100 fmol in the reaction. Furthermore, human Pol incorporated a C or a T opposite the AAF-guanine with similar efficiencies, extending 64% and 65% primers, respectively (Fig. 6B, lanes 4 and 5). Less frequently, human Pol also incorporated an A opposite the AAF-guanine, with 40% primer extension (Fig. 6B, lane 3). Least frequently, human Pol incorporated a G opposite the AAF-guanine, with 11% primer extension (Fig. 6B, lane 6). These results show that human Pol is capable of translesion synthesis opposite an AAF-adducted guanine in an error-prone manner.
Error-free lesion bypass opposite a (–)-trans-anti-benzo[a]pyrene-N2-dG lesion by human Pol
Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) is a metabolite of benzo[a]pyrene, representing an ultimate carcinogen of the parent compound. The racemic anti-BPDE reacts with DNA mainly at the N2 position of guanine, forming stereoisomeric bulky adducts (+)-trans-anti-BPDE-N2-dG, (+)-cis-anti-BPDE-N2-dG, (–)-trans-anti-BPDE-N2-dG and (–)-cis-anti-BPDE-N2-dG (43,44). 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. 7). As shown in Figure 7 (lane 1), human Pol efficiently bypassed the (–)-trans-anti-BPDE-N2-dG lesion at a low polymerase concentration (20 fmol). To identify the nucleotide incorporated opposite the lesion, we performed lesion bypass assays with only one deoxyribonucleoside triphosphate. As shown in Figure 7 (lanes 2–5), human Pol incorporated a C opposite the template (–)-trans-anti-BPDE-N2-dG. These results demonstrate that human Pol is capable of error-free lesion bypass opposite a template (–)-trans-anti-BPDE-N2-dG.
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Human Pol
is unable to bypass a TT dimer or a TT (6-4) photoproductCyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts are the major DNA lesions following UV radiation. Recently, it was discovered that DNA Pol
is capable of error-free lesion bypass opposite a TT dimer (a CPD lesion), but is unable to bypass a TT (6-4) photoproduct (16,17,27). Like Pol, Pol is also a member of the UmuC superfamily of proteins. Thus, it is of particular interest to examine how human Pol would respond to a TT dimer and a TT (6-4) photoproduct in DNA. To do this, we labeled a 15mer primer at its 5'-end with 32P and annealed it to a 49mer DNA template containing a site-specific TT dimer or a TT (6-4) photoproduct (Fig. 8). Lesion bypass assays were then carried out with purified human Pol. As shown in Figure 8 (lanes 3 and 4), human Pol was unable to insert a nucleotide opposite the 3' T of either the TT dimer or the TT (6-4) photoproduct, even at a high polymerase concentration (200 fmol). In contrast, purified human Pol efficiently bypassed the template TT dimer (Fig. 8, lane 2), as expected. These results show that human Pol is unable to bypass a TT dimer or a TT (6-4) photoproduct.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have examined the ability of the purified human Pol
in translesion DNA synthesis opposite a template 8-oxoguanine, AP site, AAF-adducted guanine, (–)-trans-anti-BPDE-N2-dG, TT dimer and TT (6-4) photoproduct. Human Pol effectively bypassed the first four DNA lesions in vitro. Furthermore, purified human Pol bypassed 8-oxoguanine, AP site, AAF-adducted guanine in an error-prone manner, but bypassed (–)-trans-anti-BPDE-N2-dG in an error-free manner. Hence, we conclude that human Pol is a novel lesion bypass DNA polymerase.
Human Pol can incorporate either a C or an A opposite 8-oxoguanine, consistent with the miscoding property of this lesion (41). However, unlike yeast Pol that predominantly incorporates a C opposite a template 8-oxoguanine (18,45), human Pol incorporates an A more frequently. Thus, the miscoding property of 8-oxoguanine is modulated by the interaction between the lesion and the specific DNA polymerase.
In a study by Johnson et al. (30), it was concluded that human Pol is unable to bypass an AP site. However, in another study by Ohashi et al. (29), it was concluded just the opposite. It should be noted that while Johnson et al. (30) used a fusion human Pol containing at its N-terminus a bulky (26 kDa) glutathione S-transferase (GST) protein, Ohashi et al. (29) used a truncated human Pol missing 310 amino acid residues at the C-terminus. Our results using a full-length human Pol are consistent with those of Ohashi et al. (29). The different results of Johnson et al. (30) with respect to AP site bypass might be due to the fused GST protein and/or lower concentrations of Pol used. Human Pol was used at 0.5 nM by Johnson et al. (30), whereas 2 nM and 4 nM were used by us (Fig. 4A) and Ohashi et al. (29), respectively.
The efficiency and specificity of nucleotide incorporations by human Pol opposite an AP site are strongly influenced by the template base 5' to the lesion. Nucleotide incorporation occurs most efficiently when the template base 5' to the AP site is a T. Nevertheless, A is most frequently incorporated opposite the AP site by human Pol, which is most dramatic when the template base 5' to the lesion is either a T or a C. Further extension by human Pol is greatly stimulated by a template T 5' to the AP site. This stimulation is largely due to a one-nucleotide deletion mechanism. We propose that following incorporation of A opposite the AP site, further extension by human Pol is efficiently achieved through misaligning the primer terminal A with the next template T, leading to –1 deletion. Supporting this model, in the absence of the 5' template T, primer extension by human Pol is largely stopped after incorporating one nucleotide opposite the AP site. Moreover, this model is also consistent with the observation that the E.coli DinB is prone to cause –1 deletion mutations (6). A similar model was also independently proposed by Ohashi et al. (29).
In E.coli, translesion synthesis of an AP site results in preferential incorporation of an A opposite the lesion, leading to the ‘A rule’ hypothesis (46). In mammals, such a strong bias opposite an AP site was not obvious. Opposite an AP site, incorporations of A, C and T at similar frequencies were reported in simian COS cells using a plasmid mutagenesis system (47–50). In another study in COS cells, preferential A incorporation was observed (51). In a study using human lymphoblastoid cells, preferential G incorporation was noticed (52).
In the yeast S.cerevisiae, the Pol–REV1 bypass of AP site results in predominantly C insertion opposite the lesion (53), while yeast Pol incorporates G and less frequently A opposite the AP site (18). However, this yeast does not contain the DINB1 gene. Humans contain Pol encoded by the REV3 (9,10) and REV7 (13) genes, REV1 (11,12) and Pol (26,27). Thus, in addition to Pol, AP sites may also be bypassed by the Pol–REV1 pathway and the Pol mechanism in human cells. Indeed, human REV1 efficiently incorporates a C opposite a template AP site in vitro (11) and purified human Pol is capable of incorporating an A and less efficiently a G opposite the template AP site (21,22). Possible involvement of multiple mechanisms and the sequence context effect during AP site bypass in mammals may provide an explanation to reconcile different results of AP site bypass by different studies.
The bulky AAF adduct blocks many DNA polymerases (18,42). However, human Pol is able to effectively bypass an AAF-adducted guanine in vitro at a high polymerase concentration (100 fmol) and incorporates C, T, less frequently A, and occasionally G, at least in the sequence context tested. Similar results were also obtained by Ohashi et al. (29) with a C-terminal truncated form of human Pol. Using a shuttle vector containing a site-specific AAF-guanine, Shibutani et al. (54) observed that A, T and G were misincorporated opposite the lesion in simian kidney cells, resulting in eight G
T, six GA and two GC mutations, respectively, from 156 clones analyzed. Our in vitro results are in general agreement with this in vivo study. Thus, Pol may contribute to AAF mutagenesis in mammals. Previously, we have shown that yeast Pol specifically incorporates a C opposite an AAF-adducted guanine (18). More recently, error-free bypass of AAF-adducted guanine has been extended to human Pol (our unpublished data) (22). AAF bypass by Pol may account for the fact that the majority of recovered shuttle vector from COS cells did not contain mutations opposite the lesion (54).
Benzo[a]pyrene is a potent environmental carcinogen. The racemic anti-BPDE compounds are cellular metabolites of benzo[a]pyrene, which readily attack DNA at the N2 position of guanine (43,44). One of the four stereoisomeric guanine adducts of racemic anti-BPDE is the (–)-trans-anti-BPDE-N2-dG (43,44). This lesion is mutagenic in COS cells (55). In this study, we observed that human Pol is capable of efficient error-free bypass opposite a template (–)-trans-anti-BPDE-N2-dG lesion at a low polymerase concentration. This result suggests that Pol may play an important role in suppressing benzo[a]pyrene-induced mutagenesis in humans.
In contrast, human Pol is unable to bypass a TT dimer and a TT (6-4) photoproduct, which are among the major lesions induced by UV radiation. On the other hand, human Pol efficiently catalyzes error-free translesion synthesis opposite the template TT dimer (17,27). Without functional Pol (encoded by the XPV gene in humans), XPV disease is manifested in humans (26,27), which is characterized by sun sensitivity and a predisposition to skin cancer (33). The dramatic phenotype of XPV patient suggests that error-free bypass of TT dimers and perhaps additional CPD lesions is mainly contributed by Pol and could not be substituted by other DNA polymerases in vivo. The inability of human Pol to bypass a TT dimer supports this conclusion.
The biological consequence of an unrepaired DNA lesion is determined by whether it is bypassed or not, bypassed in an error-free or error-prone mode and the specificity of mutations induced during lesion bypass. Since the same lesion can be responded to very differently by different DNA polymerases, it seems that the biological effect of a lesion can be significantly affected by the unique interactions between the lesion and a specific DNA polymerase. Based on this notion, it appears that the structure of a lesion alone would not be sufficient to precisely predict the biological outcome of the lesion in DNA. With the identification of Pol as a lesion bypass polymerase in vitro, it is very likely that multiple lesion bypass mechanisms exist in humans in response to DNA damage during replication.
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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.), an NIH grant CA40463 (J.-S.T.) and an 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@pop.uky.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Y. Zhang, X. Wu, O. Rechkoblit, N. E. Geacintov, J.-S. Taylor, and Z. Wang Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion Nucleic Acids Res., April 1, 2002; 30(7): 1630 - 1638. [Abstract] [Full Text] [PDF]
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D. Chiapperino, H. Kroth, I. H. Kramarczuk, J. M. Sayer, C. Masutani, F. Hanaoka, D. M. Jerina, and A. M. Cheh Preferential Misincorporation of Purine Nucleotides by Human DNA Polymerase eta Opposite Benzo[a]pyrene 7,8-Diol 9,10-Epoxide Deoxyguanosine Adducts J. Biol. Chem., March 29, 2002; 277(14): 11765 - 11771. [Abstract] [Full Text] [PDF]
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S. Avkin, S. Adar, G. Blander, and Z. Livneh Quantitative measurement of translesion replication in human cells: Evidence for bypass of abasic sites by a replicative DNA polymerase PNAS, March 19, 2002; 99(6): 3764 - 3769. [Abstract] [Full Text] [PDF]
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 V. Bergoglio, C. Bavoux, V. Verbiest, J.-S. Hoffmann, and C. Cazaux Localisation of human DNA polymerase {kappa} to replication foci J. Cell Sci., January 12, 2002; 115(23): 4413 - 4418. [Abstract] [Full Text] [PDF]
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P. Gruz, F. M. Pisani, M. Shimizu, M. Yamada, I. Hayashi, K. Morikawa, and T. Nohmi Synthetic Activity of Sso DNA Polymerase Y1, an Archaeal DinB-like DNA Polymerase, Is Stimulated by Processivity Factors Proliferating Cell Nuclear Antigen and Replication Factor C J. Biol. Chem., December 7, 2001; 276(50): 47394 - 47401. [Abstract] [Full Text] [PDF]
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 Z. Wang DNA DAMAGE-INDUCED MUTAGENESIS : A NOVEL TARGET FOR CANCER PREVENTION Mol. Interv., December 1, 2001; 1(5): 269 - 281. [Abstract] [Full Text] [PDF]
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P. M. J. Burgers, E. V. Koonin, E. Bruford, L. Blanco, K. C. Burtis, M. F. Christman, W. C. Copeland, E. C. Friedberg, F. Hanaoka, D. C. Hinkle, et al. Eukaryotic DNA Polymerases: Proposal for a Revised Nomenclature J. Biol. Chem., November 16, 2001; 276(47): 43487 - 43490. [Full Text] [PDF]
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F. Boudsocq, S. Iwai, F. Hanaoka, and R. Woodgate Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic pol{eta} Nucleic Acids Res., November 15, 2001; 29(22): 4607 - 4616. [Abstract] [Full Text] [PDF]
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N. Watanabe, E. Okochi, M. Mochizuki, T. Sugimura, and T. Ushijima The Presence of Single Nucleotide Instability in Human Breast Cancer Cell Lines Cancer Res., November 1, 2001; 61(21): 7739 - 7742. [Abstract] [Full Text] [PDF]
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A. Kondo, R. Safaei, M. Mishima, H. Niedner, X. Lin, and S. B. Howell Hypoxia-induced Enrichment and Mutagenesis of Cells That Have Lost DNA Mismatch Repair Cancer Res., October 1, 2001; 61(20): 7603 - 7607. [Abstract] [Full Text] [PDF]
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 W. Zhang, P. D. Bardwell, C. J. Woo, V. Poltoratsky, M. D. Scharff, and A. Martin Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line Int. Immunol., September 1, 2001; 13(9): 1175 - 1184. [Abstract] [Full Text] [PDF]
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D. Guo, X. Wu, D. K. Rajpal, J.-S. Taylor, and Z. Wang Translesion synthesis by yeast DNA polymerase {{zeta}} from templates containing lesions of ultraviolet radiation and acetylaminofluorene Nucleic Acids Res., July 1, 2001; 29(13): 2875 - 2883. [Abstract] [Full Text] [PDF]
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J. O-Wang, K. Kawamura, Y. Tada, H. Ohmori, H. Kimura, S. Sakiyama, and M. Tagawa DNA Polymerase {kappa}, Implicated in Spontaneous and DNA Damage-induced Mutagenesis, Is Overexpressed in Lung Cancer Cancer Res., July 1, 2001; 61(14): 5366 - 5369. [Abstract] [Full Text] [PDF]
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Y. Zhang, F. Yuan, X. Wu, J.-S. Taylor, and Z. Wang Response of human DNA polymerase {{iota}} to DNA lesions Nucleic Acids Res., February 15, 2001; 29(4): 928 - 935. [Abstract] [Full Text] [PDF]
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Y. Zhang, F. Yuan, X. Wu, O. Rechkoblit, J.-S. Taylor, N. E. Geacintov, and Z. Wang Error-prone lesion bypass by human DNA polymerase {eta} Nucleic Acids Res., December 1, 2000; 28(23): 4717 - 4724. [Abstract] [Full Text] [PDF]
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Y. Zhang, F. Yuan, H. Xin, X. Wu, D. K. Rajpal, D. Yang, and Z. Wang Human DNA polymerase {kappa} synthesizes DNA with extraordinarily low fidelity Nucleic Acids Res., November 1, 2000; 28(21): 4147 - 4156. [Abstract] [Full Text] [PDF]
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R. L. Levine, H. Miller, A. Grollman, E. Ohashi, H. Ohmori, C. Masutani, F. Hanaoka, and M. Moriya Translesion DNA Synthesis Catalyzed by Human Pol eta and Pol kappa across 1,N6-Ethenodeoxyadenosine J. Biol. Chem., May 25, 2001; 276(22): 18717 - 18721. [Abstract] [Full Text] [PDF]
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Z. Livneh DNA Damage Control by Novel DNA Polymerases: Translesion Replication and Mutagenesis J. Biol. Chem., July 6, 2001; 276(28): 25639 - 25642. [Full Text] [PDF]
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