Mammalian Translesion DNA Synthesis across an Acrolein-derived Deoxyguanosine Adduct

PARTICIPATION OF DNA POLYMERASE eta  IN ERROR-PRONE SYNTHESIS IN HUMAN CELLS*

In-Young YangDagger , Holly MillerDagger , Zhigang Wang§, Ekaterina G. Frank, Haruo Ohmori||, Fumio Hanaoka**, and Masaaki MoriyaDagger DaggerDagger

From the Dagger  Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, the § Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, the  Section on DNA Replication, Repair and Mutagenesis, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2725, the || Laboratory of Genetic Information Analysis, Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan, and the ** Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan

Received for publication, December 9, 2002, and in revised form, January 23, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha -OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying the error-free and error-prone translesion DNA syntheses, in vitro primer extension experiments using purified DNA polymerases and site-specific alpha -OH-PdG were conducted. The results suggest the involvement of pol eta  in the cellular error-prone translesion synthesis. Experiments with xeroderma pigmentosum variant cells, which lack pol eta , confirmed this hypothesis. The in vitro results also suggested the involvement of pol iota  and/or REV1 in inserting correct dCMP opposite alpha -OH-PdG during error-free synthesis. However, none of translesion-specialized DNA polymerases catalyzed significant extension from a dC terminus when paired opposite alpha -OH-PdG. Thus, our results indicate the following. (i) Multiple DNA polymerases are involved in the bypass of alpha -OH-PdG in human cells. (ii) The accurate and inaccurate syntheses are catalyzed by different polymerases. (iii) A modification of the current eukaryotic bypass model is necessary to account for the accurate bypass synthesis in human cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

During the last several years, many new DNA polymerases (pol)1 have been discovered in prokaryotes and eukaryotes (1-3). Several of these polymerases, such as eukaryotic pol eta , pol kappa , pol iota , pol zeta , and REV1 and Escherichia coli pol IV and pol V, are thought to be involved in translesion DNA synthesis. With the exception of pol zeta , which belongs to the B family, they share extensive sequence homology and comprise a new polymerase family designated the Y family (4). These polymerases are different from replicative polymerases in several aspects, i.e. they replicate more efficiently across altered bases and catalyze both accurate and inaccurate translesion DNA syntheses, they have more flexible and larger catalytic pockets (5-7) that give them the ability to tolerate damaged template bases, and they show reduced fidelity when copying unmodified DNA (8-14). Their ability to catalyze translesion synthesis has been studied extensively in vitro using various DNA lesions as substrates, but knowledge of their roles in translesion synthesis in mammalian cells is still very fragmentary. Among these polymerases, pol eta , which is defective in cells of xeroderma pigmentosum variant (XPV) patients, was shown to catalyze accurate and efficient translesion synthesis across certain UV photoproducts (15), whereas human pol zeta  (16) and REV1 (17) are involved in inaccurate syntheses across UV photoproducts. One recent study using pol kappa -defective mouse cells has shown that the enzyme is involved in the error-free translesion synthesis across a benzo[a]pyrene-dG adduct(s) (18). Two eukaryotic translesion synthesis pathways have been proposed (19-23). In one pathway, both insertion and extension steps are catalyzed by one DNA polymerase. In the other pathway, extension is catalyzed by a DNA polymerase, such as pol zeta  or pol kappa , which is different from the one inserting a nucleotide opposite a DNA lesion.

In this research, we conducted translesion synthesis studies in vitro and in vivo to probe the cellular bypass mechanism for an acrolein-derived dG adduct. Acrolein, the simplest member of the alpha ,beta -unsaturated aldehyde family, is widely found in the environment and is also produced endogenously. It initiates urinary bladder carcinogenesis in rats (24) and is mutagenic in bacteria (25, 26) and cultured cells (27-29). Acrolein reacts with dG residues in DNA to form two pairs of stereoisomeric exocyclic propano adducts (Fig. 1), namely the 8R and 8S isomers of 3H-8-hydroxy-3-(beta -D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (gamma -OH-PdG) and the 6R and 6S isomers of 3H-6-hydroxy-3-(beta -D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (alpha -OH-PdG). gamma -OH-PdG predominates over alpha -OH-PdG (30-32) and has been detected in DNA isolated from human and animal tissue (30, 33). Lipid peroxidation is suspected to be the major endogenous source (30). Comparative genotoxic studies with a site-specific adduct in human cells have shown that gamma -OH-PdG is less blocking than is alpha -OH-PdG (34) and is bypassed with high fidelity (34, 35). alpha -OH-PdG, on the other hand, miscodes substantially in human cells with a frequency of 10-12% per bypass synthesis, with Gright-arrowT being predominant (34). As alpha -OH-PdG strongly inhibits DNA synthesis (34), it is likely that the translesion polymerases are involved in bypassing this adduct. This leads to the following questions: (i) which translesion polymerase is responsible for the correct and incorrect syntheses; and (ii) whether these syntheses are catalyzed by one polymerase or by different polymerases. Here, we show the following. (i) Multiple DNA polymerases are involved in the bypass synthesis. (ii) Pol eta  participates in incorrect synthesis. (iii) The current eukaryotic bypass model (19-23) does not seem to account for the error-free bypass of this adduct.


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Fig. 1.   Structures of acrolein-derived alpha - and gamma -OH-PdG.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oligonucleotides-- The procedures for the synthesis, purification, and characterization of oligonucleotides containing alpha -OH-PdG have been described (37). The 13-mer (5'-CTCCTCXATACCT-3') and 28-mer (5'-CTGCTCCTCXATACCTACACGCTAGAAC-3'), in which X represents alpha -OH-PdG, were the same oligonucleotides as those used in our previous study (34). The 13-mer and 28-mer were used in the translesion synthesis studies in vivo (human cells) and in vitro, respectively. The 16-mer (5'-GTTCTAGCGTGTAGGT-3'), 18-mer (5'-GTTCTAGCGTGTAGGTAT) and 19-mer (5'-GTTCTAGCGTGTAGGTATN-3', in which N stands for A, G, C, or T) were employed as primers in the experiments of read-through nucleotide incorporation opposite alpha -OH-PdG and primer extension from a terminus opposite alpha -OH-PdG, respectively. The 28-mer template contained the entire sequence of the 13-mer. All unmodified as well as modified oligonucleotides were purified by electrophoresis in denaturing 20% polyacrylamide gel and formed a single band following purification.

DNA Polymerases and Proliferating Cell Nuclear Antigen (PCNA)-- Human Pol eta  (38), pol kappa  (39), pol iota  (13), REV1 (40), calf thymus pol delta  (41), and Saccharomyces cerevisiae pol zeta  (19) were purified as described. The 3'right-arrow5' exonuclease (exo)-proficient Klenow enzyme was obtained from New England BioLabs (Beverly, MA); human PCNA was a gift from Paul A. Fisher (State University of New York, Stony Brook, NY).

Primer Extension Reaction-- The 28-mer template and a 5'-32P-end-labeled primer were mixed at a molar ratio of 1:2, heated at 70 °C for 5 min, and annealed by slow cooling. Reaction mixtures (10 µl) contained 40 mM bis-Tris (pH6.8), 6 mM MgCl2, 10 mM dithiothreitol (DTT), 40 µg/ml bovine serum albumin (BSA), and 14 ng/µl PCNA for pol delta ; 40 mM Tris-HCl (pH 8.0), 30 mM KCl, 5 mM MgCl2, 10 mM DTT, and 250 µg/ml BSA for pol eta  and pol kappa  (42); 25 mM KH2PO4 (pH 7.0), 5 mM MgCl2, 5 mM DTT, 100 µg/ml BSA and 10% glycerol for REV1 (43) and pol zeta  (19); 40 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10 mM beta -mercaptoethanol (replacing DDT used in the original buffer), 250 µg/ml BSA and 2.5% glycerol for pol iota  (44); and 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 7.5 mM DTT for the Klenow enzyme. The final concentration of dNTP was 10 µM for incorporation experiments and 100 µM each in extension and read-through experiments. A primed template was added at a concentration of 40 nM. The amounts of polymerases added are indicated in the legends to Figs. 3-7. Reactions with pol delta  were incubated at 30 °C for 30 min, and those with the other enzymes were at 37 °C for 10 min. Following reaction, 7 µl of a formamide dye mixture (95% formamide, 0.1% xylene cyanol, 0.1% bromphenol blue, and 20 mM EDTA) was added, and aliquots (4 µl) were subjected to electrophoresis in denaturing (8 M urea) 20% polyacrylamide gel at 2300 V for 2.5 h. Radioactive bands were detected and, if necessary, quantified by a PhosphorImager and ImageQuant software (Amersham Biosciences).

Cell Lines-- The SV40-transformed human XPV cell lines CTag (45) and XP30RO(sv) (46) were obtained from M. Cordeiro-Stone (University of North Carolina, Chapel Hill, NC) and J. Cleaver (University of California, San Francisco, CA), respectively. CTag and XP30RO(sv) were established from XP4BE and XP30RO (GM3617), respectively. XP4BE and XP30RO cells contain a four-nucleotide (positions 289-292) and a 13-nucleotide (positions 343-355) deletion, respectively, in the coding region of one allele of the XPV gene and produce severely truncated proteins due to the new stop codons generated (47, 48). The other allele is not transcribed in either cell line. Cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (100 µg/ml), and streptomycin (100 µg/ml) at 37 °C in 5% CO2. An expression vector containing a mouse XPV cDNA (mXPV) was constructed as follows. A NotI fragment containing mXPV was isolated from pGEM-mXPV (49) and cloned in the correct orientation into the NotI site of pIRESneo2 (Clontech), which has the G418 resistance gene. The construct, pIRES-mXPV, was introduced into CTag cells by the FuGENE6 method (Roche Molecular Biochemicals) according to a manufacturer's protocol. Transfected cells were selected for G418 (Mediatech, Herndon, VA) resistance at 500 µg/ml medium. pIRESneo2 is designed to translate a cloned gene and the G418 resistance gene from the same transcript. As this transcript contains an internal ribosome entry site between the cloned gene and the G418 resistance gene, the mXPV gene and the G418 resistance gene are independently translated. Furthermore, translation of the G418 resistance gene is designed to be less efficient than that of the cloned gene. Therefore, all G418-resistant cells are expected to express mXPV. To further assure the collection of mXPV-expressing cells, G418-resistant cells were irradiated with UV at 2J/m2 and then cultured in the presence of 1 mM caffeine (49). Almost all cells transfected with the empty pIRESneo2 vector died after 4 days, whereas cells transfected with pIRES-mXPV survived. Following two cycles of this phenotypic selection, surviving cells were used as the host for site-specific experiments. Finally, the transcription of the mXPV gene was confirmed by RT-PCR (reverse transcriptase-polymerase chain reaction) using RNeasy Mini Kit (Qiagen) and SuperScript One-Step RT-PCR Kit (Invitrogen).

Translesion Synthesis Studies in Human Cells-- The shuttle vector, pBTE, was described previously (35). This vector is stably maintained in human cells and confers blasticidin S resistance to host human and E. coli cells. Expression of the resistance gene is driven by the SV40 early promoter in human cells and the EM7 bacterial promoter in E. coli. The construction of double-stranded DNA plasmid containing site-specific alpha -OH-PdG has been described (34) and is shown in Fig. 2 together with the experimental strategy. alpha -OH-PdG was incorporated into the leading strand template. An important feature of this construct is that the adduct was inserted opposite a unique SnaBI site (5'-TACGTA-3') with mismatches on both sides of the adduct (Fig. 2); thus, only the unmodified complementary strand contains the SnaBI site. Progeny plasmids derived from the unmodified strand and excision repair events are sensitive to SnaBI digestion, whereas those derived from translesion synthesis are not. Hence, progeny derived from translesion synthesis can be selectively collected for fidelity analysis by digesting with SnaBI prior to E. coli transformation.


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Fig. 2.   Outline of experimental procedure (A), construction of modified plasmid (B), and oligonucleotide probes used for sequence analysis of progeny plasmid (C). In panels B and C, note mismatches at the sequence of 3'-AXC (highlighted). X represents alpha -OH-PdG. Probe S (overscored) hybridizes only to unmodified strand. Probes L and R detect plasmid containing a 13-mer insert. Probes G, T, A, C, and D determine targeted events. Delta , single base deletion.

CTag/pIRES and CTag/pIRES-mXPV cells were seeded at 1 × 106 cells/25-cm2 flask, cultured overnight, then transfected overnight with 1 µg of a DNA construct by the FuGENE6 method. Where indicated, cells were treated with mitomycin C at 1 µg/ml medium for 50 min in an incubator, after which the medium was replaced with a fresh medium, and transfection was begun immediately. The next day, cells were detached by treating with trypsin-EDTA and replated in a 75-cm2 flask. The following day, blasticidin S (Invitrogen) was added to the culture medium at 5 µg/ml. Resistant cells were collected after 5 or 6 days. The progeny plasmid was purified by the method of Hirt (50) and treated with DpnI (2 units) for 1 h to remove residual input DNA.

To establish the apparent efficiency of translesion DNA synthesis, DpnI-treated plasmid was used to transform E. coli. To determine coding events at the site of alpha -OH-PdG, the DpnI-treated plasmid was digested with SnaBI prior to transformation. One-tenth to one-fifth of the recovered plasmid was electroporated into E. coli DH10B ElectroMAX (25 µl) (Invitrogen) by an E. coli Pulser (Bio-Rad), after which 975 µl of YT (2×) medium (36) was added, and the bacteria were cultured for 40 min at 37 °C. Portions of the transformation mixture were plated onto YT (1×) plates containing blasticidin S (50 µg/ml) and ampicillin (100 µg/ml). After overnight incubation, E. coli transformants were subjected to differential oligonucleotide hybridization (51, 52) to analyze for mutations in the adducted region. This method permits the detection of specific sequences using oligonucleotide probes. G, T, A, C, and D probes (Fig. 2C) determine coding specificity at the site of alpha -OH-PdG. The S probe hybridizes to the complementary SnaBI-containing strand. L and R probes confirm the presence of the 13-mer insert. Automated DNA sequence analysis was performed as necessary.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To understand the mechanism of the translesion synthesis across alpha -OH-PdG in human cells, we first conducted in vitro experiments to select candidate polymerases whose translesion synthesis activity and fidelity are consistent with the in vivo results, and we then examined the role of one (pol eta ) of the candidates in human cells.

Pol delta -catalyzed Translesion Synthesis-- A running start experiment (Fig. 3) using a 16-mer primer and a 28-mer template showed that pol delta  bypassed alpha -OH-PdG very weakly only in the presence of PCNA. Extended products were not observed opposite the adduct, and the majority of the extension was terminated at one base before the adduct site. These results suggest that nucleotide insertion opposite alpha -OH-PdG and the subsequent extension are poor. When the read-through experiment was catalyzed by exo+ Klenow enzyme, full-length products were rarely observed, and some extended products were observed opposite the adduct. These results suggest that the full-length products observed in the pol delta -catalyzed reaction were generated by true bypass synthesis across the adduct. No stable insertion of a nucleotide opposite alpha -OH-PdG by pol delta  was confirmed by nucleotide incorporation experiments using 10 and 100 µM dNTP (Fig. 4). The results of these experiments indicate that pol delta /PCNA catalyzes bypass of alpha -OH-PdG very weakly. At this time the fidelity of this bypass synthesis is not known.


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Fig. 3.   Translesion synthesis catalyzed by pol delta /PCNA or exo+ Klenow enzyme. 32P-5'-end-labeled 16-mer primer/28-mer template complex (40 nM) was incubated with various amounts of calf thymus pol delta  or exo+ Klenow enzyme in the presence of 100 µM each of four dNTPs at 30 °C for 30 min for pol delta  and 37 °C for 10 min for Klenow. Where indicated, PCNA (140 ng) was added to 10-µl reaction mixture. Reaction products were analyzed in denaturing 20% polyacrylamide gel. X indicates the position of alpha -OH-PdG. The activity of pol delta  decreased during prolonged storage but was not re-determined, and the original unit value was used.


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Fig. 4.   Incorporation of a nucleotide opposite alpha -OH-PdG by pol delta /PCNA. 32P-5'-end-labeled 18-mer primer/28-mer template complex (40 nM), 0.75 units of pol delta , 140 ng of PCNA, and 10 µM (left) or 100 µM (right) dNTP were used. The other conditions were the same as those in Fig. 3.

In subsequent experiments designed to determine the nucleotide distance between the adduct and the primer terminus at which pol delta /PCNA recovered efficient synthesis, we found that exonucleolytic proofreading prevailed over polymerization when the primer terminus was located three nucleotides or less 5' to the adduct (Fig. 5). When the terminus was five nucleotides away, net polymerization efficiency increased. At seven nucleotides, proofreading became marginal, and polymerization was predominant. Therefore, if a translesion polymerase catalyzes DNA synthesis >= 7 nucleotides past alpha -OH-PdG, the subsequent synthesis can be performed efficiently by pol delta .


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Fig. 5.   Resumption of DNA synthesis by pol delta /PCNA. 32P-5'-end-labeled primers of various lengths (19-26) were annealed to a modified 28-mer template, and the primer extension reaction was performed using pol delta  (0.75 units) and PCNA (140 ng) as described in the legend to Fig 3. X represents alpha -OH-PdG.

Translesion DNA Polymerase-catalyzed Bypass Synthesis-- As alpha -OH-PdG inhibits DNA synthesis strongly, it is conceivable that translesion polymerases participate in bypassing this adduct. To determine which polymerase(s) plays a role in the accurate and inaccurate bypass syntheses, we first examined the abilities of pol eta  and pol kappa  to catalyze a bypass synthesis. The running start experiments revealed that both polymerases could bypass this adduct (Fig. 6). Qualitative nucleotide incorporation experiments (Fig. 7A) showed that pol eta  inserted predominantly dAMP and, weakly, dGMP and dTMP opposite alpha -OH-PdG, whereas it preferentially inserted dCMP and, moderately, dAMP and dTMP opposite dG. The extension experiments (Fig. 7B) with the 19-mer primer revealed that dA and dG but not dC or dT termini were extended from opposite alpha -OH-PdG. These results suggest that the bypass synthesis catalyzed by pol eta  predominantly results in a Gright-arrowT transversion, which is the major miscoding event observed in human cells (34). A similar analysis with pol kappa  (Fig. 7A) showed that this polymerase inserted dGMP weakly, dAMP and dTMP marginally, and no dCMP opposite alpha -OH-PdG, whereas it predominantly inserted correct dCMP opposite dG. Extension experiments (Fig. 7B) showed that dA, dG, and dT termini, but not a dC terminus, were extended weakly. These results suggest that pol eta - and pol kappa -catalyzed bypass syntheses are inaccurate and do not account for the accurate synthesis in human cells.


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Fig. 6.   Translesion syntheses catalyzed by pol eta  and pol kappa . 32P-5'-end-labeled 16-mer primer/28-mer template complex (40 nM) was incubated with various amounts of a DNA polymerase in the presence of 100 µM each of four dNTPs at 37 °C for 10 min. Reaction products were analyzed in denaturing 20% polyacrylamide gel. X indicates the position of alpha -OH-PdG.


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Fig. 7.   Translesion DNA polymerase-catalyzed nucleotide incorporation opposite alpha -OH-PdG (A) and extension from termini opposite alpha -OH-PdG (B). A, 32P-5'-end-labeled 18-mer primer/28-mer template complex (40 nM) was incubated with a DNA polymerase in the presence of 10 µM of one dNTP at 37 °C for 10 min. Concentrations of polymerases were 3.63 nM pol eta , 3.1 nM pol kappa , 2.5 nM pol iota , 14.4 nM REV1, and 5.4 nM pol zeta  in a 10-µl reaction mixture. B, 5' 32P-labeled 19-mer primer/28-mer template complex (40 nM) was incubated with a DNA polymerase in the presence of four dNTPs (100 µM each) at 37 °C for 10 min. Concentrations of DNA polymerases were the same as those used in panel A.

In experiments using pol iota , REV1 and pol zeta  (Fig. 7, A and B), REV1 exclusively inserted correct dCMP, and pol iota  incorporated dCMP and dTMP opposite alpha -OH-PdG (Fig. 7A). However, no extension from these termini was observed (Fig. 7B), suggesting that these two polymerases require another DNA polymerase for the subsequent extension to complete accurate translesion synthesis. As pol zeta  is known to have this capability, i.e. extension of a primer from a mismatched terminus and from a terminus opposite DNA lesions (19, 53-56), we examined a pol zeta -catalyzed extension from four termini opposite alpha -OH-PdG. Although this polymerase catalyzed extension from all four termini opposite dG, with a dC terminus being most efficiently extended, the extension from a dC terminus opposite alpha -OH-PdG was much less efficient than that from the other three (dA, dG, and dT) termini (Fig. 7B). This result suggests that pol zeta  does not efficiently complete the accurate synthesis initiated by pol iota  or REV1. Rather, it may contribute to error-prone syntheses by extending from dA, dG, and dT termini generated by other polymerases. No efficient extension was observed as expected when REV1 and pol zeta  were simultaneously added to a reaction mixture (data not shown). Pol zeta  did not efficiently insert any nucleotide opposite alpha -OH-PdG (Fig. 7A). These results suggest that the combination of pol iota  and pol zeta  or REV1 and pol zeta  does not account for the accurate translesion synthesis observed in cells.

The Role of Pol eta  in Mutagenic Bypass in Human Cells-- The results of the in vitro experiments have suggested that pol eta  and pol kappa  contribute to the cellular miscoding events. Using the XPV cell line CTag, we found a lower miscoding frequency of 1.1% (Table I) than in XPA cells (10-12%) (34). However, as this XPV cell line and the XPA cell line do not have an isogenic background, it may not be appropriate to compare the results directly. To address this issue, we introduced into CTag cells an expression plasmid containing mXPV, which was previously shown to complement the defect in human XPV cells (49). The introduction of the mXPV plasmid significantly (2.4-fold, p < 0.001) increased the miscoding frequency, which was largely ascribed to the increase in the number of Gright-arrowT transversions. A similar enhancing effect (2.3-fold, p < 0.001) of mXPV was noted when control and mXPV-transfected cells were pretreated with mitomycin C. Pretreatment of cells with mitomycin C appears to cause a slight increase in miscoding frequencies in these engineered XPV cells, though the increases were not statistically significant. The fractions of progeny derived from the modified strand were 24 and 23% for CTag/pIRES and CTag/pIRES-mXPV, respectively, without mitomycin pretreatment, and 27 and 22% for CTag/pIRES and CTag/pIRES-mXPV, respectively, with pretreatment, showing no significant differences between these two cell lines. Subsequently, another XPV cell line, XP30RO(sv), was used to confirm the result with CTag; a very low miscoding frequency was also noted in this cell line (Table I). Taken together, our results indicate the following. (i) Pol eta  does not play a major role in translesion synthesis across alpha -OH-PdG. (ii) Pol eta  is not critical to error-free bypass. (iii) Pol eta  is primarily responsible for inaccurate translesion synthesis (alpha -OH-PdGright-arrowT). The latter two ideas are supported by the results of in vitro experiments (Fig. 7, A and B).


                              
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Table I
Coding events induced by alpha -OH-PdG in XPV and complemented XPV cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Acrolein is a bifunctional agent that reacts with the 1 and N2 positions of dG to form two exocyclic propano adducts. The exocyclic rings are formed in the region involved in Watson-Crick hydrogen bonding to dC. Both adducts inhibit DNA synthesis, and alpha -OH-PdG miscodes in human XPA cells (34). To investigate the cellular translesion synthesis mechanism for alpha -OH-PdG, we conducted experiments in vitro with purified eukaryotic DNA polymerases and compared the results with the previous in vivo data to deduce a likely in vivo mechanism.

The Role of Pol eta  in Inaccurate Synthesis in Human Cells-- We showed that pol eta  bypassed alpha -OH-PdG (Fig. 6), incorporated dAMP but not dCMP opposite this adduct (Fig. 7A), and extended the primer efficiently from this dA terminus (Fig. 7B). These results suggest that pol eta -catalyzed synthesis can be highly inaccurate, resulting in alpha -OH-PdGright-arrowT transversions and that pol eta  does not contribute to error-free translesion synthesis. The miscoding frequencies obtained in the two XPV cell lines (Table I) were significantly lower than those obtained in XPA cells (34), and the lowered frequencies were complemented, though not perfectly, by the introduction of mXPV (Table I). The mXPV did not affect translesion synthesis efficiency. These results indicate that pol eta  plays a minor role in the overall process of translesion synthesis but is largely responsible for the inaccurate synthesis. The involvement of pol eta  in inaccurate replication was also reported recently for gamma -OH-PdG (57). In S. cerevisiae, pol eta  has been shown to be responsible for the accurate synthesis past 8-oxo dG (58), the inaccurate synthesis past (6-4) thymine-thymine dimers (59), and both accurate and inaccurate syntheses past acetylaminofluorene dG adducts (59).

The Role of Other Translesion Polymerases in Inaccurate Synthesis in Human Cells-- We observed miscoding events in XPV (CTag) cells, though at reduced frequencies, suggesting that another polymerase(s) catalyzes inaccurate translesion synthesis in the absence of pol eta . Among the polymerases examined, we found that pol kappa  and pol iota  incorporate incorrect nucleotides opposite alpha -OH-PdG (Fig. 7A); pol kappa  bypassed this adduct (Fig. 6) and extended from the dA, dG, and dT termini (Fig. 7B); and pol iota  inserted dCMP and dTMP opposite alpha -OH-PdG (Fig. 7A), but no further extension was observed from these termini (Fig. 7B). This extension may be catalyzed by pol zeta  (Fig. 7B), as has been observed in vitro for abasic sites (53, 55, 56) and (6-4) thymine-thymine dimers (19, 54, 55).

What Mechanism Operates in Error-free Translesion Synthesis?-- The experiments confirm and extend our previous work (34), demonstrating that accurate translesion synthesis of alpha -OH-PdG is the major event in human cells, accounting for ~90% of the products. It appears unlikely that pol eta  or pol kappa  contribute to a substantial degree for the following reasons. (i) Neither polymerase inserted correct dCMP opposite alpha -OH-PdG (Fig. 7A), and neither catalyzed extension from a dC terminus opposite this adduct (Fig. 7B). (ii) XPV cells conducted error-free translesion synthesis (Table I) with a substantial level of translesion synthesis. (iii) The introduction of mXPV did not enhance the level of translesion synthesis. In contrast to pol eta  and pol kappa , pol iota  and REV1 incorporated dCMP relatively efficiently opposite alpha -OH-PdG (Fig. 7A), but extension from this dC terminus was not observed with either polymerase. Extension may be catalyzed by other polymerases such as pol zeta  and pol kappa , as has been observed for (6-4) thymine-thymine dimers (19, 54, 55) and abasic sites (53, 55, 56). Pol zeta , however, catalyzed limited extension from a dC terminus opposite alpha -OH-PdG as compared with that from the other three termini (Fig. 7B). We did not observe any fully extended products by the simultaneous addition of REV1, which exclusively inserted dCMP, and pol zeta  to a reaction mixture. Thus, the 3'-terminal dC paired to alpha -OH-PdG was very resistant to extension by pol zeta  as well as by pol kappa . It is likely, then, that pol zeta  is not involved in the accurate synthesis, but rather may play a role in inaccurate synthesis. With all the translesion polymerases examined, extension from purine (dA and dG) termini appears to be more efficient than it is from pyrimidine (dT and dC) termini, and a dC terminus is most resistant to such extension (Fig. 7B). In conclusion, pol iota  and REV1 can serve to insert dCMP, but a polymerase that can catalyze a >= 7 nucleotide extension is required to propose a two polymerase-catalyzed bypass mechanism (19-23). Thus, the mechanism for this error-free synthesis is currently unknown.

Pol delta  is possibly responsible for the accurate synthesis, though its in vitro bypass ability does not seem sufficient to account for the in vivo bypass synthesis. Another possibility is that other DNA polymerases, such as pol theta  (60), pol lambda  (60), and pol µ (60) catalyze this accurate translesion synthesis in cells. For example, our preliminary experiments have shown that pol beta , unlike translesion polymerases, extends a primer efficiently from a dC terminus (data not shown). We should also consider, however, that the current in vitro system lacks critical accessory factors that mediate the activity of these polymerases. In conclusion, our results indicate that multiple DNA polymerases are involved in the translesion synthesis across alpha -OH-PdG and that accurate and inaccurate translesion syntheses are catalyzed by different polymerases.

    ACKNOWLEDGEMENTS

We thank A. P. Grollman for his encouragement and support, F. Johnson and M. C. Torres for synthesizing modified oligonucleotides, R. Woodgate and P.A. Fisher for generous gifts of pol iota  and PCNA, respectively, and M. Cordeiro-Stone and J. E. Cleaver for providing XPV cell lines.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health, United States Public Health Service Grants CA76163 (to M. M.), CA47995 (to A. P. G.), and CA92528 (to Z. W.), and grants from the Core Research for Evolution Science and Technology, Japan Science and Technology Corporation (CREST, JST) and the Bioarchitect Research Project of RIKEN (to F. H.).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 Dagger To whom correspondence should be addressed. Tel.: 631-444-3082; Fax: 631-444-7641; E-mail: maki@pharm.sunysb.edu.

Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M212535200

    ABBREVIATIONS

The abbreviations used are: pol, DNA polymerase; alpha -OH-PdG, the 6R and 6S isomers of 3H-6-hydroxy-3-(beta -D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one; BSA, bovine serum albumin; DTT, dithiothreitol; exo, 3'right-arrow5' exonuclease; gamma -OH-PdG, the 8R and 8S isomers of 3H-8-hydroxy-3-(beta -D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one; PCNA, proliferating cell nuclear antigen; XPV, xeroderma pigmentosum variant; mXPV, mouse XPV cDNA.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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