Nucleic Acids Research, 2000, Vol. 28, No. 21 4147-4156
© 2000 Oxford University Press
Human DNA polymerase 
synthesizes DNA with extraordinarily low fidelity
Graduate Center for Toxicology and 1Department of Chemistry, University of Kentucky, Lexington, KY 40536, 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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Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase
(Pol) encoded by the DINB1 gene. Our recent studies have indicated that human Pol is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Pol also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Pol during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Pol synthesizes DNA with extraordinarily low fidelity. At the lacZ
target gene, human Pol made on average one error for every 200 nucleotides synthesized, with a predominant T
G transversion mutation at a rate of 1/147. The overall error rate of human Pol is 1.7-fold lower than human Pol
, but 33-fold higher than human Polß, a DNA polymerase with very low fidelity. Thus, human Pol is one of the most inaccurate DNA polymerases known. These results support a role for human Pol in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mutagenesis is a key factor to evolution, human hereditary diseases and cancer. It is now clear that mutagenesis is an active biological response to genotoxic stress conditions, such as cellular exposure to radiations and many chemical carcinogens. Following DNA damage, the resulting mutagenesis is divided into two categories depending on sites of occurrence: targeted mutagenesis and untargeted mutagenesis. The former induces mutations opposite a DNA lesion; while the latter induces mutations in undamaged regions of DNA. Untargeted mutagenesis normally refers to the mutagenic consequence of exogenously induced DNA damage, but at genomic sites without the actual lesion, including a physically separated chromosome, phage or virus that had never been damaged. Untargeted mutagenesis differs from spontaneous mutagenesis in that the latter is the combined end point of errors of the cellular replication machinery and mutagenesis opposite background DNA damage. Spontaneous mutagenesis occurs in the absence of exogenous DNA damaging agents.
In Escherichia coli, both targeted mutagenesis and untargeted mutagenesis are largely under the control of the SOS system that can be switched on by DNA damaging agents. Targeted mutagenesis is mainly mediated by the UmuCD pathway, also known as the damage-induced mutagenesis pathway (1,2). DNA polymerase (Pol) V composed of the UmuCD'2 protein complex is responsible for the translesion DNA synthesis step (3–5). Untargeted mutagenesis requires DNA polymerase IV, encoded by the dinB gene (6). Artificially overexpressing the dinB gene in E.coli is sufficient to trigger untargeted mutagenesis without exogenous DNA damage (7). Both targeted mutagenesis and untargeted mutagenesis empower E.coli cells the genomic flexibility, i.e. accelerated genetic changes in the presence of genotoxic stress conditions such as DNA damage.
In eukaryotes, the REV3 mutagenesis pathway is believed to play an important role in targeted mutagenesis. Originally discovered in the yeast Saccharomyces cerevisiae, this mutagenesis pathway has been recently recognized to also function in humans (8–12). The REV3 mutagenesis pathway involves multiple proteins, including REV1. REV1 possesses a dCMP transferase activity capable of inserting a dCMP opposite a template apurinic/apyrimidinic (AP) site (10,13). The role of REV1 in targeted mutagenesis opposite other DNA lesions remains to be elucidated. Yeast Rev1, yeast Rad30, E.coli UmuC and E.coli DinB form the prototypic members of the UmuC superfamily (14,15). RAD30 codes for DNA Pol in eukaryotes (16). In humans, Pol is encoded by the xeroderma pigmentosum variant (XPV) gene, whose inactivation leads to the hereditary XPV disease (17,18). A role for Pol in both error-free and error-prone lesion bypasses has been shown recently by in vitro studies (16,17,19–22). The in vivo function of human Pol in error-free lesion bypass following UV radiation has been unequivocally established by the phenotypes of XPV patients (23). A human homologue of the E.coli dinB has been identified, DINB1 (24,25). Human DINB1 protein is a DNA polymerase (26,27) and is designated Pol by several laboratories (15,26,28). Our recent in vitro studies show that human Pol is capable of both error-free and error-prone translesion DNA synthesis (28). Therefore, it appears that proteins of the UmuC superfamily are involved in various mechanisms of lesion bypass in response to unrepaired DNA damage during replication.
Most recently, the eighth S.cerevisiae DNA polymerase was identified as the TRF4 gene product and was named Pol (29), which is required for sister chromatid cohesion. Our analysis by protein sequence alignment indicates that this polymerase has no relation to the human Pol encoded by the DINB1 gene. In fact, this yeast does not contain a sequence homologue of the BINB1 gene.
Much less is known about untargeted mutagenesis in eukaryotes. However, there are indications that untargeted mutagenesis does occur in eukaryotes including mammals. For example, after replicating shuttle vectors containing a site-specific AAF in COS cells, untargeted mutagenesis was observed surrounding the lesion (30,31). Since human Pol is capable of translesion DNA synthesis in vitro (26,28), the possibility is raised that Pol may be involved in untargeted mutagenesis surrounding a lesion during lesion bypass by this polymerase. Furthermore, overexpression of mouse DINB1 gene in mouse cells results in elevated mutagenesis (25), implicating Pol in a more general mechanism of untargeted mutagenesis.
To gain insights into the possible role of Pol in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage, we have measured the fidelity of human Pol during DNA synthesis from undamaged templates. In this report, we show that human Pol synthesizes DNA with extraordinarily low fidelity, thus supporting a role for Pol in untargeted mutagenesis in humans.
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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 Klenow fragment of E.coli DNA polymerase I was from BRL (Bethesda, MD). The yeast rad30 deletion mutant strain BY4741rad30
(MATa his3 leu2 met15 ura3 rad30) was purchased from Research Genetics (Huntsville, AL). Human testis cDNA and human placenta cDNA were purchased from Clontech Laboratories (Palo Alto, CA). Gapped M13mp2 DNA (32) for initial studies was provided by Thomas Kunkel (National Institute of Environmental Health Sciences, Research Triangle Park, NC). Escherichia coli strains MC1061 and CSH50 were provided by Dale Mosbaugh (Oregon State University, Corvallis, OR).
Plasmid constructions
The human DINB1 cDNA was obtained by polymerase chain reaction (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 yeast expression vector pECUh6 (28), yielding pECUh6–hDINB1. The human XPV cDNA was obtained by PCR amplification from human testis cDNAs using Pfu DNA polymerase and two primers: 5'-CGGGATCCATGGCTACTGGACAGGATCGAG-3' and 5'-ACGCGTCGACCATTGT-ACCCGGCCGAG-3'. The resulting 2.6 kb PCR product was then cloned into the BamHI and SalI sites of the yeast expression vector pECUh6 (28), yielding pECUh6–XPV. The human POLB cDNA was obtained by PCR amplification from human placenta cDNAs using Pfu DNA polymerase and two primers: 5'-GCTCTAGAATGAGCAAACGGAAGGCGC-3' and 5'-CCCAAGCTTCCCATTGGGTTGTGTCTGCG-3'. The resulting 1.1 kb PCR product was then cloned into the XbaI and HindIII sites of the yeast expression vector pEGUh6 (33), yielding pEGUh6–hPOLB.
Purification of human Pol, Pol and Polß
To express human Pol and Pol, yeast rad30 deletion mutant cells containing pECUh6–hDINB1 or 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 medium (2% Bacto-peptone, 1% yeast extract, 2% dextrose), cells were grown for 6 h at 30°C. Expression of the human genes was induced by adding CuSO4 to 0.3 mM and growth for another 3 h. To express human Polß, yeast SX46A (MATa ade2 his3-532 trp1-289 ura3-52) cells harboring pEGUh6–hPOLB were grown in minimal medium containing 2% sucrose for 2 days. Expression of Polß was induced by diluting the culture 10-fold in 16 l of YPG medium (2% Bacto-peptone, 1% yeast extract, 2% galactose) supplemented with 0.5% sucrose and incubation for 15 h at 30°C with shaking. Collected cells 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 (34,35). 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 Pol, Pol or Polß were identified by western blots using a mouse monoclonal antibody specific to the His6 tag.
To purify Pol and Pol further, the pooled nickel column samples were 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 samples (40 ml each) were loaded onto a Mono S HR5/5 column (Amersham Pharmacia Biotech) separately and eluted with a 30 ml linear gradient of 100–400 mM KCl in FPLC buffer A. Both Pol and Pol were eluted at 250 mM KCl. Fractions containing Pol or Pol were pooled and concentrated by PEG 10 000. The samples were then loaded separately onto an FPLC Superdex 200 gel filtration column equilibrated with FPLC buffer A containing 300 mM KCl and eluted in the same buffer.
To purify human Polß further, the nickel column fractions were concentrated and desalted as described above. Then, the sample was loaded onto an FPLC Resource S column (1 ml) and eluted using the same buffer system and gradient as described in Pol and Pol purifications. Polß was eluted at 230 mM KCl. The Resource S fractions were concentrated and loaded onto an FPLC Superdex 200 column using the same method as in Pol and Pol purifications. Polß was eluted at 40 kDa position.
DNA polymerase assays
A standard DNA polymerase reaction (10 µl) contained 25 mM KH2PO4 pH 7.0, 5 mM MgCl2, 5 mM dithiothreitol, 100 µg/ml bovine serum albumin, 50 µM each of dATP, dCTP, dTTP and dGTP, 50 fmol of a primed DNA template, 10% glycerol and a purified DNA polymerase. 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). Reaction products were separated by electrophoresis 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.
Kinetic measurements of DNA polymerases
Kinetic analyses of human DNA polymerases were performed as previously described (36,37). Briefly, DNA polymerase assays were performed using 50 fmol of a primed DNA template, 0.7 ng (7 fmol) of purified human Pol and increasing concentrations of each dNTP (dATP, dCTP, dTTP or dGTP). The DNA primer, 5'-GGAAGAAGAAGTATGTT-3', was labeled at its 5'-end with 32P. The four DNA templates used were: template A, 5'-CCTTCTTCATTCAAACATACTTCTTCTTCC-3'; template G, 5'-CCTTCTTCATTCGAACATACTTCTTCTTCC-3'; template C, 5'-CCTTCTTCATTCCAACATACTTCTTCTTCC-3'; and template T, 5'-CCTTCTTCATTCTAACATACTTCTTCTTCC-3', with the analyzed template base underlined. After incubation for 10 min at 30°C under standard DNA polymerase assay conditions, reaction products were separated by electrophoresis on a 20% denaturing polyacrylamide gel. The percentage of primers extended by the polymerase was calculated following scanning densitometry of the extended DNA band(s) and the remaining primer band on the autoradiogram. Product formed (P) was derived from the calculation: P = % primer extension x 50 fmol. Observed enzyme velocity (v) was obtained from the calculation: v = P/10 min. Then, the observed enzyme velocity was plotted as a function of the dNTP concentrations. The plotted data was fitted by a non-linear regression curve to the Michaelis–Menten equation, v = (Vmax x [dNTP])/(Km + [dNTP]), using the SigmaPlot software. Vmax and Km of a DNA polymerase for the incorporation of the correct and the incorrect nucleotides were obtained from the fitted curve. Relative polymerase selectivity of incorrect versus correct nucleotides (finc) was finally calculated from the equation: finc = (Vmax/Km)incorrect/(Vmax/Km)correct (37).
M13mp2 DNA synthesis fidelity assays
DNA synthesis reaction mixture (10 µl) contained 25 mM KH2PO4 (pH 7.0), 5 mM dithiothreitol, 100 µg/ml bovine serum albumin, 30% glycerol, 100 µM dNTP, 8 fmol of gapped M13mp2 DNA (407 nt gap expanding into the lacZ gene) and 400 fmol of purified human Pol, 205 fmol of purified human Pol or 1200 fmol of purified human Polß. To completely fill the gap, excess amounts of DNA polymerases were needed (32), especially for human Polß, due to the distributive mode of DNA synthesis by these polymerases. After incubation at 30°C for 1 h, the reaction was terminated by mixing 2 µl of the reaction mixture with 2 µl of 40 mM EDTA. The remaining reaction mixture (8 µl) was analyzed by electrophoresis on a 0.8% agarose gel to monitor the gap filling reaction. Then, the terminated reaction mixture was diluted 5-fold in water and 1 µl was transfected by electroporation into competent cells (50 µl) of E.coli strain MC1061. In order to detect mutant M13mp2 phage plaques, the transfected cells were plated on plates containing IPTG, X-gal and a lawn of E.coli strain CSH50 as described by Bebenek and Kunkel (32). M13mp2 phage plaques were developed following incubation of the plates at 37°C overnight. Mutant M13mp2 phage plaques (light blue and colorless) were picked and plaque-purified before DNA sequencing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of human Pol
, Pol and PolßPurified human Pol
was used to study its DNA synthesis fidelity in vitro. For comparison, human Pol and Polß were also purified. To facilitate the purification of human Pol, Pol and Polß, we tagged all three proteins with six histidine residues at their N-termini and expressed them in the yeast S.cerevisiae cells. The yeast rad30 deletion mutant cells were used for Pol and Pol expression to avoid potential contamination by the yeast Pol. The three human DNA polymerases were purified separately to apparent homogeneity (Fig. 1). The identity of these purified polymerases were confirmed by western blot analyses using a mouse monoclonal antibody specific to the His6 tag (data not shown). The three polymerases were biochemically active as tested by DNA polymerase assays (data not shown) (28). Human Pol and Polß migrated as 77 and 42 kDa proteins, respectively, consistent with their calculated molecular weights of 78 and 38 kDa, respectively (Fig. 1B and C). Human Pol migrated as an 81 kDa protein on a 10% SDS–polyacrylamide gel, faster than its calculated molecular weight of 99 kDa (Fig. 1A). However, there are indications suggesting that human Pol migrated abnormally faster than predicted from its molecular weight (28). Thus, our purified human Pol, Pol and Polß are apparently the full-length proteins.
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Human Pol
does not possess a 3'5' proofreading nuclease activityTo examine whether Pol
contains a proofreading nuclease activity, we labeled several primers at their 5'-ends with 32P and annealed them to a 30mer DNA template (Fig. 2A). These DNA substrates contained a TC mismatch (Fig. 2A, primer P1), an AC mismatch (Fig. 2A, primer P2), a CT mismatch (Fig. 2A, primer P3) or a GA mismatch (Fig. 2A, primer P4) at the primer 3'-end. In the absence of dNTPs, a 3'5' proofreading nuclease would recognize a mismatch at the primer 3'-end and subsequently remove one or a few nucleotides from the 3'-end of the primer. Under the conditions used, the proofreading nuclease activity of the Klenow fragment of E.coli DNA polymerase I was readily detected after 2 min incubation (Fig. 2B, compare lanes 1 and 2). In contrast, human Pol did not degrade the mismatched primers even after 30 min incubation in the absence of dNTPs (Fig. 2C). These results show that human Pol does not possess a 3'5' proofreading nuclease activity.
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Nucleotide incorporations by human Pol
To examine the base pairing property during DNA synthesis catalyzed by human Pol
, we performed DNA polymerase assays in the presence of only one deoxyribonucleoside triphosphate using 7 fmol of Pol and 50 fmol of primed 30mer DNA templates. A 17mer primer was labeled with 32P at its 5'-end and annealed to templates C, G, T and A (Fig. 3A). Opposite the template CC sequence, two G residues were incorporated by Pol as the major event (Fig. 3B, lane 5). Less frequently, T and A were also incorporated at a decreasing rate (Fig. 3B, lanes 4 and 2). Opposite the template T, A was predominantly incorporated (Fig. 3B, lane 7). Less frequently, C, G and T were also incorporated (Fig. 3B, lanes 8–10). Opposite the template G, a C residue was predominantly incorporated by Pol (Fig. 3C, lane 3). Less frequently, T, G and A were also incorporated at a decreasing rate (Fig. 3C, lanes 4, 5 and 2). Opposite the template A, T was predominantly incorporated by Pol (Fig. 3C, lane 9) and, although to a much lesser extent, G was also incorporated (Fig. 3C, lanes 10). In the presence of all four dNTPs as controls, human Pol efficiently extended the primers near the end of the templates (Fig. 3B and C, lanes 1 and 6). It was consistently observed that human Pol stopped DNA synthesis one or two nucleotides before the end of the template (Fig. 3B and C, lanes 1 and 6) (27,28). These results show that human Pol preferentially incorporates the correct base opposite the template C, T, G or A, but is prone to misincorporations.
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Kinetic measurements of base selection by human Pol
To obtain a quantitative measurement of base selections by human Pol
, we measured its nucleotide incorporation fidelity using a steady state kinetic analysis (36,37). DNA polymerase assays were performed with increasing concentrations of a single dNTP using 50 fmol of a primed 30mer DNA template (Fig. 3A) and 7 fmol of purified human Pol. Opposite the template G, correct C incorporation was observed at very low dCTP concentrations (Fig. 4A, lanes 2–7). However, for T misincorporation, primer extension occurred at much higher dTTP concentrations (Fig. 4A, lanes 8–14). These base incorporation results were quantitated and the observed incorporation velocities (v) were calculated. Experimental v values were plotted against the dNTP substrate concentrations and fitted into the Michaelis–Menten equation, v = (Vmax x [dNTP])/(Km + [dNTP]), as shown in Figure 4B. From the fitted curve, the kinetic parameters Vmax and Km were obtained and listed in Table 1.
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Using this method, we systematically measured the Vmax and Km values of all 16 possible base incorporations opposite the four template bases (Table 1). As indicated by the Vmax/Km values (Table 1), template T was most likely to lead to misincorporations, whereas template A was least likely to lead to misincorporations. Based on the Vmax/Km results (Table 1), G and C misincorporations opposite template T occurred most frequently; while C misincorporation opposite template C and A misincorporation opposite template A occurred least frequently. Discrimination between right and wrong nucleotides as indicated by finc (Table 1) show that human Pol
significantly misincorporated nucleotides opposite template A, G, C and T. These kinetic measurements suggest that human Pol is a low fidelity DNA polymerase.
Mismatch extension by human Pol
Mutation formation during normal DNA synthesis is determined by two steps: (i) misincorporation followed by (ii) mismatch extension by the DNA polymerase. To determine if human Pol is able to extend misincorporated bases, we performed mismatch extension assays using 40 fmol of the purified polymerase and 50 fmol of a primed DNA template containing a mismatch at the primer 3'-end. All 12 possible mismatches were examined (Fig. 5A). As shown in Figure 5B, at the polymerase concentration used, all mismatches were extended by human Pol, but with very different efficiencies. While G–G, G–T and T–C (template–primer) mismatches were extended relatively efficiently, A–A mismatch was extended with least efficiency (Fig. 5B). In comparison, a Watson–Crick base pair such as T–A was extended most efficiently by human Pol (Fig. 5B, lane 26).
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In most cases, primer extension by human Pol
stopped one or two nucleotides before the end of the template, regardless of correct base pair or mismatch at the primer 3'-end prior to extension (Fig. 5B, lanes 2, 6, 8, 10, 14, 20, 22, 24 and 26). With T–T and A–G mismatches, however, significant amounts of the primers were extended one nucleotide shorter than others (23mer DNA band) (Fig. 5B, lanes 4 and 12). The 23mer extension products were probably derived from –1 misalignment prior to extension, as the primer T could pair with the next template A in the T–T mismatch substrate and the primer G could pair with the next template C in the A–G mismatch substrate (Fig. 5A). Consistent with this interpretation, a tendency for E.coli DNA polymerase IV (DinB protein) to make –1 template–primer misalignment has been observed (6,7). With C–T and C–C mismatches, several shorter bands were observed (Fig. 5B, lanes 16 and 18), which might result from multiple and/or various template–primer misalignments. These results show that human Pol can readily extend some mismatched base pairs, although not as efficiently as extending a correct base pair; and suggest that some mismatch extensions may be mediated by the template–primer misalignments.
DNA synthesis fidelity of human Pol
To determine if frequent misincorporations and the ability of mismatch extension by human Pol would indeed lead to a high mutation rate, we measured the fidelity of this polymerase in synthesizing 229 nt of the target lacZ gene (from nucleotide –84 to +145) (32). For comparison, we also measured the fidelity of purified human Pol and Polß. To perform the fidelity assay, a gapped M13mp2 DNA was prepared containing a 407 nt gap (32). Within the gap lies the lacZ template DNA strand. The gap was then filled in by purified human Pol in a standard DNA polymerase reaction buffer. Complete gap filling by human Pol slowed the DNA migration to an identical position as the nicked circular M13mp2 control DNA on a 0.8% agarose gel (compare Fig. 6A and B). Pol and Polß also completely filled in the gap in M13mp2 DNA (Fig. 6C and D). After gap filling by the purified polymerases, the phage DNA was then transfected into the E.coli MC1061 cells for mutant identification. In the presence of IPTG and X-gal, wild-type M13mp2 phage produced dark blue plaques, whereas mutants were identified as light blue or colorless phage plaques (32). Human Pol yielded a mutation frequency of 30% at the lacZ target gene, which was similar to human Pol (49%), but considerably higher than human Polß (2.7%) (Table 2).
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To identify the specific mutations during DNA synthesis by human Pol
from the undamaged template, we isolated 53 mutant M13mp2 phage clones and sequenced the target gene. Within the 229 nt target sequence, 50 of the 53 mutant clones contained multiple mutations, up to 8 mutation sites per target sequence and 4 tandem double-base substitutions were observed. On average, 3.8 mutation sites per target sequence were produced by Pol (Table 2). The overall error rate of Pol is thus one mutation for every 200 nucleotides synthesized (3.8 x 3.0 x 10–1/229 = 5.0 x 10–3 = 1/200). For comparison, we also sequenced 53 Pol- and 32 Polß-induced mutants. Pol produced 3.9 mutations per target sequence (Table 2). In contrast, Polß produced 1.3 mutations per target sequence, as expected (Table 2). The overall error rate of Pol is 1.7-fold lower than human Pol, but 33-fold higher than human Polß. Thus, human Pol and Pol are two of the most inaccurate DNA polymerases known, synthesizing DNA with unprecedented high error rates.
Major mutations produced by Pol and Pol were base substitutions, followed by deletions (Table 2). Most deletion mutations were single base deletions. One nucleotide deletion mutations of –T, –G, –C and –A (on the DNA +strand) were scored 5, 4, 2 and 2 times, respectively, among the 204 total mutations at the lacZ target sequence. One nucleotide insertion mutations of +A, +G, +C and +T (on the DNA +strand) were scored 2, 2, 1 and 1 times, respectively. Additionally, one mutant lacZ sequence contained a stretch of 73-nt deletion and a stretch of 16-nt insertion. These results show that human Pol synthesizes DNA with extraordinarily low fidelity.
Specificity of base substitution mutations by human Pol
Base substitution error rates by human Pol are summarized in Table 3. Based on the total number of mutations produced opposite template A, G, C and T (base substitutions in Table 3 plus deletions and insertions), template bases were copied by human Pol with very different fidelity and followed the order from most accurate to most inaccurate: A > C > G > T. All 12 possible base substitutions were observed. As a result of C mispairing with template T, TG transversion was the most frequent mutation event during DNA synthesis by Pol with an error rate of 1/147 (Table 3). The second highest base substitution error rate observed was TC transition as a result of G mispairing with template T (Table 3). The third highest base substitution error rate observed was GA transition as a result of T mispairing with template G (Table 3). The lowest base substitution error rate (1/10 000) observed was AC transversion as a result of G mispairing with template A (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using three different approaches, we found that purified human Pol
synthesizes DNA with extraordinarily low fidelity. In the first approach, we examined base misincorporations by human Pol opposite template C, T, G and A under the standard polymerase assay conditions. Significant misincorporations were observed. In the second approach, we measured kinetic parameters of base incorporations by human Pol. These two methods yielded consistent results, indicating that human Pol significantly incorporates incorrect bases, especially opposite a template T, C or G. Furthermore, both approaches show that human Pol is catalytically most efficient opposite a template G. In the third approach, we determined the fidelity of human Pol during in vitro synthesis of a 229 nt lacZ sequence. This method reflects the ability of Pol to incorporate incorrect bases and to subsequently extend mismatches. It detects both point and frameshift mutations (32). Low fidelity DNA synthesis of human Pol was again clearly demonstrated.
Human Polß has been recognized as a low fidelity DNA polymerase (38). Remarkably, human Pol is 33-fold more inaccurate than human Polß. Human Pol generates on average one mutation for every 200 nucleotides synthesized, while human Pol generates on average one mutation for every 120 nucleotides synthesized. Extraordinarily low fidelity of human Pol has also been reported most recently by Matsuda et al. (39). Hence, human Pol and Pol are in the same category of unprecedented low fidelity DNA polymerases. Although the overall error rates between human Pol and Pol are similar, the mutation specificity generated by these two polymerases is quite different. While the most frequent base substitution mutations observed with human Pol is TG (C incorporation opposite template T), followed by TC (G incorporation opposite T), the most frequent base substitution mutations observed with human Pol is TC, followed by AT (A incorporation opposite A) (our unpublished data) (39).
Overexpressing the E.coli dinB gene in E.coli cells results in higher spontaneous mutations, mostly –1 deletions (7). Surprisingly, deletions were not the major mutations produced during DNA synthesis by human Pol, accounting for only 9.3% of the total mutations, which is much lower than 87% of base substitutions. It would be very informative to examine the intrinsic fidelity of E.coli DNA polymerase IV using the M13mp2 DNA synthesis fidelity assay and compare to human Pol. In the M13mp2 fidelity assay, due to the distributive mode of DNA synthesis by human Pol, Pol and Polß, these polymerases were used in excess amounts relative to DNA in order to completely fill in the 407-nt gap. Nevertheless, the error rates of base substitutions observed in this assay (Table 3) are in good agreement with the error rates of base misincorporations (Table 1) obtained by kinetic measurements where much lower polymerase to DNA ratio was used. Thus, it is unlikely that excess amounts of the polymerase used in the M13mp2 fidelity assay could drastically affect the intrinsic fidelity of the polymerase. Supporting this conclusion, Matsuda et al. (39) observed similar mutation frequencies of purified human Pol during synthesis of the M13mp2 DNA gap at several polymerase concentrations.
Most recently, Tang et al. (4) examined base misincorporations by E.coli polymerase IV using steady state kinetic analyses. It was reported that E.coli polymerase IV incorporated nucleotides 5–10-fold more accurately than E.coli polymerase V (4). Since the reported finc for E.coli polymerase IV ranged mostly from 10–4 to 10–5 (4), the result would predict that this polymerase is not remarkably error-prone, which seems to be inconsistent with the in vivo results of untargeted mutagenesis by polymerase IV (7,40). Most recently, using kinetic analysis, Johnson et al. (27) also examined nucleotide miscincorporations by purified human DINB1 protein. Our results significantly differ from those of Johnson et al. (27). For example, it was reported that G incorporation opposite template A was the most frequent misincorporation (27). Our kinetic measurements indicate that this incorporation is among the least frequent misincorporation and our M13mp2 fidelity assays show that mutations resulting from this misincorporation represent the lowest base substitutions. In general, kinetic measurements of Johnson et al. (27) predict human Pol to be a significantly more accurate DNA polymerase than what we have observed. The precise basis for this discrepancy is presently unknown. While our human Pol contained a short N-terminal His6 tag, the human DINB1 protein isolated by Johnson et al. (27) contained at its N-terminus a bulky GST protein (26 kDa) and the E.coli polymerase IV used by Tang et al. (4) contained at its N-terminus a bulky maltose binding protein (42.7 kDa). This raises the possibility that the fused bulky proteins may have somewhat altered the properties of the human Pol and the E.coli DNA polymerase IV.
Both Pol and Pol are members of the recently identified UmuC superfamily of proteins (14,15). The important role of Pol in response to UV radiation has been well demonstrated by the phenotypes of the human hereditary disease XPV (23). The role of Pol in human biology, however, is not well understood. Our biochemical studies indicate that human Pol is a novel lesion bypass enzyme. Purified human Pol is capable of error-free bypass opposite a template (–)-trans-anti-benzo[a]pyrene-N2-dG bulky lesion and error-prone bypass opposite a template 8-oxoguanine, abasic site and AAF-adducted guanine (28). It is conceivable that lesion bypass DNA polymerases may contain a loose and flexible active site pocket to accommodate damaged DNA templates for translesion DNA synthesis. Consequently, lesion bypass polymerases may have lost stringent constraints at their active sites for high fidelity Watson–Crick base pairings. This hypothesis predicts that all lesion bypass polymerases are associated with a very low DNA synthesis fidelity. Supporting this hypothesis, the lesion bypass enzymes Pol and Pol of humans are associated with extraordinarily low DNA synthesis fidelity. Further supporting the hypothesis, human Pol
is able to incorporate one nucleotide opposite several DNA lesions, notably a template abasic site (41); and this polymerase is found to be highly error-prone on undamaged DNA templates (41,42).
Due to the high error rate of human Pol, lesion bypass by this polymerase may expose the surrounding DNA sequences to a much higher probability of mutations leading to untargeted mutagenesis. Lesion bypass by Pol could not only gain survival for the cell, but also may empower the cell to evolve and adapt through mutations at both the targeted and untargeted sequences. Additionally, the highly error-prone nature of human Pol also supports a role for this polymerase in untargeted mutagenesis in undamaged regions of DNA. Indeed, overexpressing the mouse DINB gene, a condition that may mimic presumptive up-regulation of the gene under genotoxic stress conditions, results in elevated spontaneous mutation rates in mouse cells (25). It is likely that the untargeted mutagenesis function of DinB protein may have been evolutionally conserved from E.coli to humans. Our results presented here and in another report (28) support two functions for human Pol: (i) DNA lesion bypass and (ii) untargeted mutagenesis.
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ACKNOWLEDGEMENTS |
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We thank Thomas Kunkel for providing us with the gapped M13mp2 DNA for initial studies. We thank Dale Mosbaugh for the E.coli strains MC1061 and CSH50. We thank Thomas Kunkel and Katarzyna Bebenek for technical advice on the M13mp2 DNA synthesis fidelity assay. This work was supported by a New Investigator Award in Toxicology from Burroughs Wellcome Fund and a THRI grant from the University of Kentucky.
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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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