Translesion Synthesis past Acrolein-derived DNA Adduct, gamma -Hydroxypropanodeoxyguanosine, by Yeast and Human DNA Polymerase eta *

Irina G. Minko, M. Todd Washington, Manorama Kanuri, Louise Prakash, Satya Prakash, and R. Stephen LloydDagger

From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555

Received for publication, July 31, 2002, and in revised form, September 9, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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gamma -Hydroxy-1,N2-propano-2'deoxyguanosine (gamma -HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol eta  can incorporate a C opposite gamma -HOPdG at ~190- and ~100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at ~8- and ~19-fold lower efficiency. Although DNA synthesis past gamma -HOPdG by yeast pol eta  was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of ~10-1 to 10-2. Because gamma -HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced gamma -HOPdG. For both yeast and human pol eta , the ring open reduced gamma -HOPdG adduct was less blocking than gamma -HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing gamma -HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol eta  might potentially contribute to both error-free and mutagenic bypass of gamma -HOPdG.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Acrolein (Fig. 1), the simplest alpha ,beta -unsaturated aldehyde, is an environmental contaminant and a product of inborn metabolism. In organisms, acrolein is generated via a number of pathways, such as the oxidation of polyamines and lipid peroxidation (1, 2). Like many other bifunctional aldehydes, acrolein reacts with DNA bases to form several DNA adducts, among which the gamma -hydroxy-1,N2-propano-2'deoxyguanosine (gamma -HOPdG)1 was identified as a major deoxyguanosine (dG) derivative (3, 4). Importantly, gamma -HOPdG has been detected in DNA from mammalian tissues (5-7), suggesting that this adduct is generated in vivo. The gamma -HOPdG adduct is formed by conjugate addition of acrolein to N2 of dG to produce N2-(3-oxopropyl)dG. Ring closure at N1 leads to the formation of the cyclic adduct (Fig. 1). In the nucleoside and presumably in single-stranded DNA, gamma -HOPdG predominantly exists in the cyclic form, such that at physiological pH, the ring open species cannot be detected spectrophotometrically (8). However, in the presence of a reducing agent, the acyclic form can be trapped as the N2-(3-hydroxypropyl) adduct (Fig. 1).


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Fig. 1.   Structures of the acrolein and related deoxyguanosine adducts.

Another dG derivative, 1,N2-propanodeoxyguanosine (PdG) (Fig. 1), whose structure is similar to that of the ring closed gamma -HOPdG, has been extensively exploited as a model compound for the gamma -HOPdG and other exocyclic dG adducts in both structural and biological studies. NMR spectroscopy of the PdG-adducted oligodeoxynucleotides has revealed that when placed opposite dC, PdG adopts a syn orientation within the duplex and introduces a localized structural perturbation that is pH- and sequence-dependent (9, 10). The inability of PdG to form normal Watson-Crick hydrogen bonds severely blocks DNA synthesis both in vitro (11, 12) and in vivo (13-16), and the replication that does occur results in mutations (13-16). Specifically, PdG-induced base substitutions occurred at an overall frequency of 7.8 × 10-2 and 7.5 × 10-2/translesion synthesis in the COS-7 (14) and in the nucleotide excision repair-deficient human cells (16), respectively. In both strains, G to T transversions predominated.

Recently, the structure of the gamma -HOPdG-containing oligodeoxynucleotide was solved by NMR spectroscopy (17). These data have indicated that within the duplex, gamma -HOPdG exists primarily in the ring open form. In such a conformation, the modified base participates in standard Watson-Crick base pairing by adopting a regular anti orientation around the glycosidic torsion angle, with the N2-propyl chain in the minor groove pointing toward the solvent (17). The structural differences between PdG and gamma -HOPdG within the duplex have led to the hypothesis that the latter lesion would be less blocking for replication and less mutagenic than the former.

Biological studies aimed to test the cytotoxic and mutagenic effects of acrolein-modified DNAs and of site-specific gamma -HOPdG adduct have generated conflicting results. It is known that acrolein itself causes mutations in both bacterial (18) and mammalian (19) systems and has tumor-initiating activity (20). When a DNA vector was treated with acrolein and propagated in human cells, the majority of mutations were single, tandem, and multiple base substitutions that predominantly occurred in G:C base pairs (21). However in bacteria, gamma -HOPdG, the major acrolein-derived dG adduct, is not a strong block for DNA synthesis nor a miscoding lesion (22-24). Analyses of mutations caused by gamma -HOPdG in wild type Escherichia coli and in polB, dinB, and umuDC deficient strains revealed that in the absence of these "SOS" polymerases, the efficiency and accuracy of the translesion synthesis were not significantly affected (22). In contrast to the prokaryotic data, gamma -HOPdG caused mutations at an overall frequency of 7.4 × 10-2/translesion synthesis when a single-stranded, site-specifically modified vector was propagated in COS-7 cells (24). Interestingly, both the frequencies and types of mutations were remarkably similar to those reported for the PdG adduct (14, 16). However, gamma -HOPdG was shown to be only marginally miscoding (<= 1% base substitution) when double-stranded vector was utilized (16). In this investigation, a number of cell lines including HeLa, a nucleotide excision repair-deficient xeroderma pigmentosum group A, and polymerase eta -deficient xeroderma pigmentosum variant were examined.

Although replication across gamma -HOPdG in vivo was predominantly error-free (from 93 to 100% of the translesional events), the adduct was shown to be a severe block and a miscoding lesion during in vitro DNA synthesis by a number of polymerases. Particularly, replication across gamma -HOPdG by the Klenow exo- fragment of E. coli polymerase I was significantly inhibited and extremely error-prone (22, 23). gamma -HOPdG also strongly blocked DNA synthesis by two major eukaryotic polymerases, pol delta  and pol epsilon  (24). In the presence of proliferating cell nuclear antigen, little bypass of the adduct by pol delta  was achieved, and it appeared to be highly mutagenic (24). We hypothesized therefore that in mammalian cells, specialized, translesion DNA synthesis polymerases (25, 26) are involved in promoting replication across gamma -HOPdG.

Among DNA polymerases proficient in translesion synthesis, yeast polymerase eta  (a product of the RAD30 gene) (27) and its human counterpart (a product of the RAD30A (XPV, POLH) gene) (28, 29) both possess a unique ability to replicate efficiently and accurately past a cis-syn cyclobutane pyrimidine dimer (30, 31), the predominant DNA lesion caused by ultraviolet irradiation. In the yeast Saccharomyces cerevisiae, deletion of RAD30 confers moderate sensitivity to UV irradiation and leads to increased UV-induced mutagenesis (32). Mutations in the human RAD30A gene cause the variant form of xeroderma pigmentosum (XPV), suggesting that predisposition of XPV individuals to sunlight-induced skin cancer is due to the lack of accurate translesion DNA synthesis across UV-induced DNA lesions (28, 29, 33). Yeast and human pol eta  also efficiently bypass a product of oxidative DNA damage, the 7,8-dihydro-8-oxoguanine, and do so in a predominantly error-free manner (34). In addition, several other DNA lesions were reported to be substrates for human (35-39) and yeast (35, 40) pol eta .

In the present study, the ability of yeast and human pol eta  to perform translesion DNA synthesis across gamma -HOPdG has been examined, and the efficiency and fidelity of synthesis have been tested using steady-state kinetic analyses. To further explore the bypass mechanism, comparative studies were also performed with two model DNA adducts: PdG, which mimics the cyclic form of gamma -HOPdG, and N2-(3-hydroxypropyl)dG, which is similar to gamma -HOPdG in its ring open form. In addition, the mutagenic potential of gamma -HOPdG was tested in vivo in both human fibroblasts and pol eta -deficient XPV cells utilizing a site-specifically modified single-stranded pMS2 vector.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- T4 DNA ligase, T4 polynucleotide kinase, and EcoRV were obtained from New England BioLabs (Beverly, MA). S1 nuclease and proteinase K were purchased from Invitrogen. [gamma -32P]ATP was purchased from PerkinElmer Life Sciences. Bio-Spin columns were purchased from Bio-Rad. Centricon 100 concentrators were obtained from Amicon Inc. (Beverly, CA). Slide-A-Lyzer Dialysis Cassettes were obtained from Pierce.

Strains and Vectors-- Single-stranded pMS2 DNA was a generous gift from Dr. M. Moriya (State University of New York, Stony Brook, NY). SV40-transformed cTAG derived from XP4BE cells and SV80 normal human fibroblasts were obtained from Dr. Marila Cordeiro-Stone (University of North Carolina, Chapel Hill, NC). The E. coli DH10B cells used for amplification of transformed DNA isolated from mammalian cells were purchased from Invitrogen.

Oligodeoxynucleotides-- 12-mer oligodeoxynucleotide modified with gamma -HOPdG, 5'-GCTAGC(gamma -HOPdG)AGTCC-3', was kindly provided by Dr. T. M. Harris and Dr. C. M. Harris (Vanderbilt University, Nashville, TN), and it was prepared by a previously described procedure (8). The 24-mer oligodeoxynucleotide, 5'-GCAGTATCGCGC(PdG)CGGCATGAGCT-3', adducted with PdG was synthesized as described (41) and was a generous gift from Dr. L. J. Marnett (Vanderbilt University, Nashville, TN). Nondamaged 12- and 24-mer with a dG in place of gamma -HOPdG or PdG, respectively, were purchased from Midland Certified Reagent Co. (Midland, TX). All of the other oligodeoxynucleotides were synthesized by the Molecular Biology Core Laboratory of the National Institute of Environmental Health Sciences Toxicology Center at the University of Texas Medical Branch (Galveston, TX) and purified by electrophoresis through a 15% denaturing PAGE (in the presence of 7 M urea).

Construction of site-specifically modified linear templates for in vitro replication assays was done according to the previously described procedure (24). Sequences of the resulting oligodeoxynucleotides were identical: 5'-GCTAGCGAGTCCGCGCCAAGCTTGGGCTGCAGCAGGTC-3', where the underlined G is either gamma -HOPdG or nonadducted dG and 5'-GCAGTATCGCGCGCGGCATGAGCTGCGCCAAGCTTGGGCTGCAGCAGGTC-3', where the underlined G is either PdG or nonadducted dG. To obtain the N2-(3-hydroxypropyl)dG-containing DNA substrate, 10 µl of 1 M NaBH4 dissolved in 1 M Hepes buffer (pH 7.4) were added twice to 200 µl of the gamma -HOPdG-adducted 38-mer oligodeoxynucleotide (1-2 µM). Each addition of the reducing agent was followed by incubation at room temperature for 4 h. DNA was then dialyzed against 10 mM Tris-HCl (pH 7.0), 1 mM EDTA overnight using Slide-A-Lyzer Dialysis Cassette (3,500 molecular weight cut off). To confirm the completeness of reduction, the polypeptide trapping technique was utilized (42) modified by A. J. Kurtz for gamma -HOPdG-containing DNAs. Briefly, probes of both gamma -HOPdG- and reduced gamma -HOPdG-adducted oligodeoxynucleotides (50 nM) were incubated with 50 mM lysine-tryptophan-lysine-lysine in the presence of 25 mM NaCNBH3 and 100 mM Hepes (pH 7.4) for 5 h. The reactions were terminated by the addition of an equal volume of 95% (v/v) formamide, 20 mM EDTA, 0.02% (w/v) xylene cyanol, and 0.02% (w/v) bromphenol blue and heating at 90 °C for 2 min. Next, DNAs were resolved through a 15% denaturing PAGE and visualized with PhosphorImager Screen. Under these conditions, no trapping was detected in reactions with gamma -HOPdG-containing oligodeoxynucleotide, whereas the gamma -HOPdG-containing DNA was completely complexed with the polypeptide.

Pol eta  Purification-- Purifications of yeast pol eta  and human pol eta  were done as described in Refs. 27 and 31, respectively.

DNA Polymerase Reaction-- The 21-mer oligodeoxynucleotides were used as primers for in vitro polymerase reactions. Their sequences were: 5'-CCTGCTGCAGCCCAAGCTTGG-3', which is complementary to the 38-mer gamma -HOPdG-containing template DNAs from positions -9 to -29 relative to the site of lesion (-9 primer) as well as complementary to the PdG-adducted 50-mer from positions -15 to -35 (-15 primer); 5'-AGCCCAAGCTTGGCGCGGACT-3' and 5'-AGCTTGGCGCAGCTCATGCCG-3', which are complementary from the position -1 to -21 to the gamma -HOPdG-containing template and the PdG-containing template, respectively (-1 primers); and 5'-GCCCAAGCTTGGCGCGGACTC-3' and 5'-GCTTGGCGCAGCTCATGCCGC-3', which overlap the lesion site in modified templates (0 primers). Primer oligodeoxynucleotides were phosphorylated with T4 polynucleotide kinase using [gamma -32P]ATP and purified using P-6 Bio-Spin columns supplied with 10 mM Tris-HCl buffer (pH 7.4). The gamma -32P-labeled primers were mixed with the oligodeoxynucleotide substrates at a molar ratio of 1:2 in the presence of 25 mM Tris-HCl buffer (pH 7.6), 50 mM NaCl, heated at 90 °C for 2 min, and cooled to room temperature overnight.

Primer extension and single-nucleotide incorporation experiments with yeast pol eta  were carried out as described (27) and with human pol eta  as in Ref. 31. Briefly, the reaction mixture (10 µl) contained 5 nM primer annealed to a template, 25 mM Tris-HCl buffer (pH 7.5), 10 mM NaCl, 5 mM MgCl2, 10% glycerol, 100 µg/ml of bovine serum albumin, 5 mM dithiothreitol, 100 µM of each of the four dNTPs (primer extension experiments), or 10 µM individually (single-nucleotide incorporation experiments), and yeast or human pol eta  at the concentrations as indicated in the figure legends. The reactions were incubated at 22 °C and terminated by the addition of 4× excess of stop solution consisting of 95% (v/v) formamide, 20 mM EDTA, 0.02% (w/v) xylene cyanol, and 0.02% (w/v) bromphenol blue. The reaction products were resolved through a 20% denaturing PAGE and visualized by a PhosphorImager screen.

Steady-state Kinetic Analysis-- Steady-state kinetic assays were carried out under the same conditions as the DNA polymerase assays except that 1 nM yeast or human pol eta  and 20 nM DNA substrates were used with various concentrations of one of the four nucleotides. The reactions were quenched after 5 min. Quantitative analyses were performed using a PhosphorImager screen and Image-Quant 5.0 software (Molecular Dynamics, Sunnyvale, CA). Calculations of rates of nucleotide incorporation were done as described in Ref. 43. The rates of nucleotide incorporation were graphed as a function of nucleotide concentration, and the kcat and Km parameters were obtained from the best fit of the data to the Michaelis-Menten equation.

Construction of Circular Single-stranded pMS2 DNA Modified with gamma -HOPdG-- The 12-mer oligodeoxynucleotides containing either gamma -HOPdG or a nondamaged dG were phosphorylated at the 5' end with ATP and inserted into single-stranded pMS2 shuttle vector as described earlier (24). The two ligated samples were designated pMS2(dG) and pMS2(gamma -HOPdG).

Mutagenesis Experiments-- Transfection of pMS2(dG) and pMS2(gamma -HOPdG) into cTAG and SV80 cells, isolation of DNA, amplification in E. coli DH10B cells, and differential hybridization analysis were done as previously described (24). Hybridization with the progeny plasmid DNA was performed using [gamma -32P]ATP-labeled 18-mer oligodeoxynucleotide probes (5'-GATGCTAGCNAGTCCATC-3', where N refers to A, T, G, or C). Whatman 541 filters containing hybridized colonies were exposed to X-Omat AR film overnight, and autoradiographs were developed to identify mutation frequency and types of mutations. Representative colonies were subjected to dideoxy sequencing (44) to confirm the presence of the mutations. A 20-mer primer (5'-CCATCTTGTTCAATCATGCG-3') sequence around 100 nucleotides downstream of the adduct was used for sequencing the region containing the 12-mer oligodeoxynucleotide in progeny plasmid DNA.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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In Vitro Lesion Bypass with Yeast DNA Polymerase eta -- To examine whether yeast pol eta  was able to replicate past a gamma -HOPdG adduct, running start primer extension experiments were performed (Fig. 2A). A 21-mer primer was annealed to the template DNA so that it allowed the addition of 9 nucleotides before encountering the adduct (-9 primer). On the nondamaged DNA substrate, primers were efficiently extended by yeast pol eta  (Fig. 2A, lanes 1-4). On the gamma -HOPdG-containing substrate (Fig. 2A, lanes 5-8), yeast pol eta  appeared to be capable of bypassing the lesion and forming full-length products. However, DNA synthesis was partially inhibited right before the DNA lesion and opposite from it.


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Fig. 2.   Primer extension (A) and single-nucleotide incorporation (B) catalyzed by S. cerevisiae pol eta  on the gamma -HOPdG-, reduced gamma -HOPdG-, and PdG-adducted templates. The 21-mer primers were annealed to the nondamaged (ND-38), gamma -HOPdG-adducted, or reduced gamma -HOPdG-adducted 38-mer templates or to the nondamaged (ND-50) or PdG-adducted 50-mer DNA templates. The DNA substrates (5 nM) were incubated at 22 °C in the presence of all four dNTPs (100 µM) with 1 nM S. cerevisiae pol eta  for a period of time as indicated (A) or in the presence of one of the four dNTPs and 4 nM S. cerevisiae pol eta  for 20 min (B). -, no nucleotide added; A, dATP; C, dCTP; G, dGTP; T, dTTP; G*, position of the modified G on the template.

To understand better the importance of ring opening during replication, primer extension experiments were carried out using two model DNA substrates: the PdG adduct, which is an analogue of the ring closed form of the gamma -HOPdG, and the reduced gamma -HOPdG, which is similar to the ring open form of the natural adduct. In the case of the 50-mer PdG-containing substrate, 21-mer primer was positioned on the template so that the incorporation of 15 nucleotides was needed before reaching the lesion (-15 primer). Because both efficiency and accuracy of the DNA synthesis are known to be sequence-dependent (43, 45), an additional nondamaged control 50-mer DNA template was utilized that had the same sequence as the PdG-adducted template. These data revealed that the PdG adduct was a much stronger block for replication by yeast pol eta  than gamma -HOPdG. Under conditions that allowed an efficient replication of the nondamaged DNA template (Fig. 2A, lanes 13-16), DNA synthesis on the PdG-adducted template was greatly inhibited one nucleotide before the lesion, and synthesis was completely aborted after incorporating a nucleotide opposite the lesion (Fig. 2A, lanes 17-20). However, replication by yeast pol eta  beyond the PdG can be achieved but at much higher concentrations of enzyme (data not shown). With the reduced gamma -HOPdG-adducted template (Fig. 2A, lanes 9-12), the bypass efficiency by yeast pol eta  seemed to be comparable with that on the gamma -HOPdG-adducted template.

The specificity of nucleotide incorporation by yeast pol eta  opposite and beyond the lesions was also tested. To identify the nucleotide that is incorporated by this polymerase opposite the adducted base, single-nucleotide incorporation experiments were carried out using standing start DNA substrates in which 3' terminus of the primer was located one nucleotide before the lesions (-1 primers) (Fig. 2B). On both nondamaged substrates, yeast pol eta  preferentially incorporated a C opposite G (Fig. 2B, lanes 3 and 18). Incorporation of a T and to a lesser extent an A and a G was also observed, especially on the 38-mer template. Interestingly, incorporation of a correct nucleotide (C) was predominant opposite each of the modified bases, namely the gamma -HOPdG (Fig. 2B, lane 8), the reduced gamma -HOPdG (Fig. 2 B, lane 13), and the PdG (Fig. 2B, lane 23) adducts.

To test whether any misincorporation occurred past the lesion site, single-nucleotide incorporation experiments were carried out using DNA substrates in which the correct nucleotide (C) was primed with the adducted base (0 primers). No nucleotide misincorporation was observed on any of the adducted templates examined (data not shown).

Thus, yeast pol eta  is capable of bypassing the gamma -HOPdG adduct, and in contrast to all other polymerases tested so far (22-24), it predominantly incorporates the correct nucleotide opposite and downstream of the lesion. In addition, these data show that a cyclic PdG is a much stronger block for replication by yeast pol eta  than an acyclic reduced gamma -HOPdG, but neither of the model adducts seem to be particularly miscoding for this polymerase.

In Vitro Lesion Bypass with Human DNA Polymerase eta -- Primer extension reactions and single-nucleotide incorporation experiments were carried out with human pol eta  (Fig. 3) using the same set of the primer/templates as with the yeast enzyme. Similar to the yeast pol eta , human polymerase was able to replicate past the gamma -HOPdG (Fig. 3A, lanes 5-8) and the reduced gamma -HOPdG lesions (Fig. 3A, lanes 9-12). However, unlike yeast pol eta , at higher enzyme concentrations human pol eta  appeared to bypass the PdG adduct (Fig. 3A, lanes 17-20).


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Fig. 3.   Primer extension (A) and single-nucleotide incorporation (B) catalyzed by human pol eta  on the gamma -HOPdG-, reduced gamma -HOPdG-, and PdG-adducted templates. The 21-mer primers were annealed to the nondamaged (ND-38), gamma -HOPdG-adducted, or reduced gamma -HOPdG-adducted 38-mer templates or to the nondamaged (ND-50) or PdG-adducted 50-mer DNA templates. The DNA substrates (5 nM) were incubated at 22 °C for 20 min in the presence of all four dNTPs (100 µM) and increased concentrations (from 0.4 to 4 nM) of human pol eta  (A) or in the presence of one of the four dNTPs and 1 nM human pol eta  (B). -, no nucleotide added; A, dATP; C, dCTP; G, dGTP; T, dTTP; G*, position of the modified G on the template.

Single-nucleotide incorporation experiments with human pol eta  revealed significant differences between the human and yeast enzymes in their discrimination abilities during nucleotide insertion opposite the gamma -HOPdG adduct. Whereas yeast pol eta  preferentially incorporated the correct nucleotide (C) opposite the lesion, human polymerase extended the -1 primer almost equally well in the presence of A, C, and G (Fig. 3B, lanes 6-10). On the PdG-adducted template, the difference between these two polymerases was even more striking. In contrast to the yeast pol eta  that incorporated a C opposite PdG, human polymerase inserted A, G, and T better than the correct nucleotide (Fig. 3B, lanes 21-25). Interestingly, incorporation by human pol eta  is much more accurate opposite the reduced gamma -HOPdG adduct (Fig. 3B, lanes 11-15) than opposite the nonreduced adduct (Fig. 3B, lanes 6-10).

Single-nucleotide incorporation experiments were carried also out using 0 primers with the C primed with the adducted base. Yielding data similar to that of the yeast pol eta , human polymerase preferentially incorporated the correct nucleotide on all five substrates tested (data not shown).

Efficiency of Nucleotide Incorporation and Extension-- To compare the efficiency of translesion synthesis by yeast and human pol eta , steady-state kinetic parameters kcat and Km were first determined for the correct nucleotide (C) incorporation opposite dG in two different sequence contexts and also opposite gamma -HOPdG, reduced gamma -HOPdG, and PdG adducts. The reactions were performed using the same 21-mer -1 primers as in the single-nucleotide incorporation experiments. For yeast pol eta , C is incorporated opposite the ring closed PdG adduct with a 1600-fold lower efficiency (kcat/Km) than C is incorporated opposite the unadducted dG (Table I). In contrast, yeast pol eta  incorporates a C opposite the ring open reduced gamma -HOPdG with only a 12-fold lower efficiency than opposite the unadducted dG. The efficiency of incorporation opposite the gamma -HOPdG adduct is in between these two extremes with a 190-fold reduction relative to the unadducted dG. The same trends were also observed with human pol eta  (Table II).

                              
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Table I
Steady-state kinetics of nucleotide incorporation by S. cerevisiae pol eta

                              
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Table II
Steady-state kinetics of nucleotide incorporation by human pol eta

Next, the steady-state kinetic parameters were determined for the extension from a C residue paired with the modified bases and were used to determine the efficiency of extending from each adduct relative to the extension from an unadducted dG (Tables I and II). For both yeast and human pol eta , the efficiencies of extensions from the gamma -HOPdG and the reduced gamma -HOPdG were reduced ~5-30-fold relative to the unadducted dG. In contrast, the extension from the PdG was blocked to a much greater extent, especially in the case of the yeast enzyme (6800-fold; Table I).

Fidelity of Nucleotide Incorporation by Yeast and Human Pol eta  Opposite gamma -HOPdG-- In the single-nucleotide incorporation experiments, yeast and human pol eta  displayed different accuracies of replication across the gamma -HOPdG adduct. To further evaluate the accuracy of nucleotide incorporation opposite the lesion, kinetic analyses were carried out using -1 primer, and the frequencies of misincorporation were calculated as the ratio of kcat/Km of the incorrect nucleotide to the correct nucleotide (43). These data showed that yeast pol eta  synthesizes past gamma -HOPdG relatively accurately with efficiency of incorporation of a C ~75 times higher than that of the next most preferred nucleotide (G) (Table I). In contrast, human pol eta  discriminated poorly between the correct and wrong nucleotides incorporating opposite gamma -HOPdG. Particularly, high misincorporation frequencies were observed for A and G (Table II).

Mutagenicity of gamma -HOPdG-modified Single-stranded pMS2 Vectors in Normal Human Fibroblasts and XPV Cells-- Table III shows the outcomes of in vivo replication of pMS2 (dG) and pMS2 (gamma -HOPdG) in SV80 and XPV cells. The data presented for XPV cells were obtained from five independent experiments. All of the 1104 E. coli transformants resulting from replication of modified pMS2 (gamma -HOPdG) in XPV cells were picked and grown in 96-well plates. Hybridization analysis revealed that 767 colonies hybridized with either one of the four probes, whereas 337 colonies did not hybridize with any of the four probes. Of those transformants that did not hybridize with any sequence-specific probe, none of those hybridized to sequences immediately upstream of the oligodeoxynucleotide ligation site, suggesting that this deletion was not caused by the adduct. Although 96% of the hybridized transformants did not contain any targeted mutations (Table III), 1.3% (10/767) were G to A transitions, 0.5% (4/767) were G to C transversions, and 2.1% (16/767) were G to T transversions. Sequencing of plasmid DNA prepared from these colonies confirmed the presence of T, C, or A, respectively, opposite the site of the adducted guanine. No mutations were observed when 192 colonies were screened out of transformants obtained from nonadducted pMS2(dG).

                              
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Table III
Single base pair substitutions in cTAG (XPV) cells and SV80 (normal human fibroblasts) transformed with single-stranded pMS2(dG) and pMS2(gamma -HOPdG)

When these experiments were repeated in SV80 normal human fibroblasts, all of the 288 transformed colonies subsequently obtained from two experiments were analyzed for mutations by differential hybridization strategy. Although only 92 colonies hybridized with either one of the four probes, 89% (82/92) contained the correct base opposite the adducted guanine, whereas 8.6% (8/92) were G to T transversions, and 1.1% (1/92) were G to C and G to A mutations. None of the colonies from the control pMS2(dG) transformants showed any mutation. Thus, XPV cells appeared to have a lower mutation frequency (3.9%) when compared with normal human fibroblast cells (11%).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The gamma -HOPdG adduct was not a significant block for replication when site-specifically modified vectors were propagated in E. coli (22-24) or in mammalian cells (16, 24). In E. coli, the adduct appeared not to be miscoding (22-24). Depending on the cell type and vector used, 93-100% of the translesion events were nonmutagenic during in vivo replication in mammalian cells (16, 24). Thus, in both prokaryotic and eukaryotic systems, DNA polymerases exist that are able to synthesize past gamma -HOPdG efficiently and in a predominantly error-free manner. On the other hand, none of the polymerases examined in vitro so far, namely, Klenow exo- fragment of E. coli pol I (22, 23), calf thymus pol delta  (24), and human pol epsilon  (24), were able to incorporate the correct nucleotide opposite this adduct. In the present study, yeast pol eta  has been identified as the first polymerase that possesses an ability to replicate across the gamma -HOPdG adduct relatively accurately. Comparable efficiency of DNA syntheses past gamma -HOPdG was also observed for human pol eta , but this polymerase displayed a much higher propensity for misincorporation. Single-nucleotide experiments as well as steady-state analyses showed that human pol eta  frequently incorporates an A or a G opposite gamma -HOPdG and therefore is likely to introduce G to T and G to C transversions.

We note that the observed kcat for C incorporation opposite the undamaged G template (~5 min-1; Table I) is slower than the rate of nucleotide incorporation measured during processive synthesis (~80 min-1; Ref. 46) for yeast pol eta . This suggests that kcat reflects the rate of DNA release and thus is an underestimate of the actual rate of nucleotide incorporation. Nevertheless, because the observed Km is expected to be decreased with the kcat in a compensatory manner, the efficiencies of nucleotide incorporation (kcat/Km) determined under steady-state conditions provide a measure of catalytic efficiencies of the enzyme. More detailed kinetic studies are needed, however, to more accurately define the mechanisms controlling the fidelity of pol eta  opposite these DNA adducts.

The nucleotide incorporation data for pol eta  are in agreement with results of the in vivo replication assays when site-specifically modified single-stranded pMS2 vector was propagated in XPV cells. Overall mutagenic frequency determined in the XPV cells (3.9 × 10-2/translesion synthesis) was about two and three times less than that in COS-7 (24) and normal human cells, respectively. Importantly, lower frequencies of transversions (particularly G to T) in XPV cells, but not G to A transitions, accounted for the observed differential between two types of cells. Thus, pol eta  might potentially contribute to the bypass of the gamma -HOPdG adduct in mammalian cells being responsible for both error-free and error-prone replicative events.

Based on the NMR spectroscopy data, a model of error-free bypass of gamma -HOPdG has been proposed in which the incoming dCTP triggers a structural rearrangement of the adduct from the ring closed to the ring open form. This change allows the formation of the standard Watson-Crick hydrogen bonds, stabilizes the structure, and facilitates the subsequent extension reaction (17). To examine the role of ring opening during replication by pol eta , we compared the efficiency of incorporation opposite gamma -HOPdG to the incorporation opposite the two model adducts: PdG and reduced gamma -HOPdG. For both yeast and human pol eta , cyclic PdG was a very strong block for the incorporation of a C relative to the acyclic reduced gamma -HOPdG. For incorporation opposite gamma -HOPdG, both polymerases had an intermediate incorporation efficiency. Ring opening was also important for the extension from a C paired with the adduct. For both yeast and human pol eta , relative efficiencies of extension were similar when gamma -HOPdG- and reduced gamma -HOPdG-modified DNA substrates were used. By contrast, the cyclic PdG adduct is a very strong block for extension by these polymerases, especially for the yeast enzyme. Overall, these data are consistent with the proposed model of de los Santos (17), such that ring opening of gamma -HOPdG is essential not only for efficient incorporation opposite the lesion by yeast and human pol eta  but also for efficient extension. However, from these data it cannot be concluded whether the incoming nucleotide causes the transformation of the adduct from the ring closed to the ring open form or whether the equilibrium is shifted toward ring open conformation by protein-DNA interactions in the polymerase active site.

The steady-state kinetic analyses and single-nucleotide incorporation experiments have revealed significant differences between yeast and human pol eta  with respect to their accuracies of replication across modified bases. For the human enzyme, frequencies of misincorporation opposite gamma -HOPdG were on average, 1 order of magnitude higher than for the yeast enzyme. In addition, the incorporation by human pol eta  opposite PdG was extremely error-prone, whereas yeast pol eta  inserted the correct nucleotide preferentially.

The proficient ability of yeast and human pol eta  to replicate across the ring open form of gamma -HOPdG strongly indicates that in spite of the fact that it is located in the minor groove, the presence of this adduct on the templating residue poses no significant hindrance to these polymerases. This suggests the lack of any specific contact of these enzymes with the minor groove of the templating residue, which would permit pol eta  to replicate across DNA adducts, which protrude into the minor groove.

Although DNA synthesis past gamma -HOPdG by pol eta  is very efficient when the adduct exists in its ring open form, in vivo replication data (16, this report) clearly show that pol eta  is not solely responsible for bypass of this lesion in humans. Thus, another polymerase is likely involved in translesion synthesis across gamma -HOPdG. The yet unidentified polymerase may be able to efficiently bypass the ring closed form of gamma -HOPdG and perhaps other exocyclic dG adducts (1, 2), in which N1 modification prevents Watson-Crick pairing.

    ACKNOWLEDGEMENTS

We acknowledge Dr. Masaaki Moriya (Department of Pharmacological Sciences, State University of New York at Stony Brook, NY) for the generous gift of pMS2 vector, Dr. Lawrence J. Marnett (Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University at Nashville, TN) for the generous gift of PdG-adducted oligodeoxynucleotide, and Dr. Marila Cordeiro-Stone (Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC) for the generous gift of XPV cells. We are grateful to Dr. Lubomir V. Nechev, Dr. Thomas M. Harris, and Dr. Constance M. Harris (Department of Chemistry, Center in Molecular Toxicology, Vanderbilt University, Nashville, TN) for synthesis of gamma -HOPdG-adducted oligodeoxynucleotide and for helpful discussions. We also acknowledge the Molecular Biology Core Laboratory at the National Institute of Environmental Health Sciences Toxicology Center (University of Texas Medical Branch, Galveston, TX) for oligodeoxynucleotide synthesis.

    FOOTNOTES

* This work was supported in part by National Institute of Health Grants ES06676 (to R. S. L.), ES00267 (to R. S. L.), and GM19261 (to L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of the Mary Gibbs Jones Distinguished Chair in Environmental Toxicology from the Houston Endowment. To whom correspondence should be addressed. Tel.: 409-772-2179; Fax: 409-772-1790; E-mail: rslloyd@utmb.edu.

Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M207774200

    ABBREVIATIONS

The abbreviations used are: gamma -HOPdG, gamma -hydroxy-1,N2-propano-2'deoxyguanosine; dG, deoxyguanosine; PdG, 1,N2-propanodeoxyguanosine; pol, DNA polymerase; XPV, xeroderma pigmentosum variant.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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