Translesion DNA Synthesis by Yeast DNA Polymerase eta  on Templates Containing N2-Guanine Adducts of 1,3-Butadiene Metabolites*

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

From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1071

Received for publication, August 28, 2000, and in revised form, October 30, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast DNA polymerase eta  can replicate through cis-syn cyclobutane pyrimidine dimers and 8-oxoguanine lesions with the same efficiency and accuracy as replication of an undamaged template. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing N2-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Here we showed that yeast polymerase eta  replicates DNA containing the monoadducts (S)-butadiene monoepoxide and (S,S)-butadiene diolepoxide N2-guanines albeit at an ~200-300-fold lower efficiency relative to the control guanine. Interestingly, nucleotide incorporation opposite the (R)-butadiene monoepoxide and the (R,R)-butadiene diolepoxide N2-guanines was ~10-fold less efficient than incorporation opposite their S stereoisomers. Polymerase eta  preferentially incorporates the correct nucleotide opposite and downstream of all four adducts, except that it shows high misincorporation frequencies for elongation of C paired with (R)-butadiene monoepoxide N2-guanine. Additionally, polymerase eta  does not bypass the (R,R)- and (S,S)-butadiene diolepoxide N2-guanine-N2-guanine intra- strand cross-links, and replication is completely blocked just prior to the lesion. Collectively, these data suggest that polymerase eta  can tolerate the geometric distortions in DNA conferred by the N2-guanine butadiene monoadducts but not the intrastrand cross-links.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Various pathways exist in cells to overcome replication blockage caused by DNA lesions. One such pathway, translesion DNA synthesis, involves specialized polymerases that, unlike replicative polymerases, are able to perform DNA synthesis on a damaged DNA template (reviewed in Refs. 1-3). Translesion DNA synthesis can be error-free or error-prone, depending on the chemical structure of the lesion and the polymerase utilized for translesion replication. Among the eukaryotic DNA polymerases, yeast and human DNA polymerases eta  perform efficient and accurate replication past a cis-syn cyclobutane pyrimidine dimer, a predominant DNA lesion formed by ultraviolet irradiation (4-7). In the yeast Saccharomyces cerevisiae, deletion of RAD30, which encodes pol eta ,1 confers moderate sensitivity to UV irradiation and an increase in UV-induced mutagenesis (8).

Mutations in the human RAD30A gene, the counterpart of the yeast RAD30, cause the variant form of xeroderma pigmentosum (9, 10). Xeroderma pigmentosum variant cells are hypermutable in response to UV irradiation, and they exhibit a significantly reduced ability to bypass a T-T dimer (reviewed in Ref. 3). Consequently, xeroderma pigmentosum variant individuals suffer from a high incidence of sunlight-induced skin cancers.

7,8-Dihydro-8-oxoguanine is one of the lesions formed by oxidative damage to DNA. Yeast and human pol eta  both efficiently bypass the 7,8-dihydro-8-oxoguanine lesion. Whereas other polymerases insert A opposite this lesion, pol eta  preferentially inserts a C (11). Thus, pol eta  is unique among DNA polymerases in its ability to bypass a T-T dimer and a 7,8-dihydro-8-oxoguanine lesion efficiently and accurately.

Here we examined the ability of yeast pol eta  to carry out translesion synthesis on DNA substrates containing N2-guanine adducts of stereoisomeric 1,3-butadiene metabolites. 1,3-Butadiene is a potent carcinogen in mice and to a lesser extent in rats (12) and has been classified as a probable human carcinogen. Butadiene-mediated carcinogenesis is initiated through its reactive metabolites: butadiene monoepoxide, butadiene diepoxide, and butadiene diolepoxide. Each of these metabolites is represented by at least two stereoisoforms. The mutagenicity of butadiene and its reactive metabolites has been observed in several biological systems, particularly in yeast (13, 14) and mammalian cells (15). Butadiene epoxides can react at numerous sites in DNA, forming a multitude of adducts that differ in their stereochemistry (16, 17). Butadiene epoxides are potent inhibitors of synthesis by DNA polymerases. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing (R)- and (S)-BDO N2-guanines and (R,R)- and (S,S)-BDE N2-guanines (18) as well as (R,R)- and (S,S)-BDE N2-guanine-N2-guanine intrastrand cross-links (19). Here we examine the action of yeast pol eta  on these two types of the N2-guanine epoxide-containing DNA substrates.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DNA Substrates with Site-specific Lesions-- The oligodeoxynucleotides containing butadiene epoxide N2-guanine adducts were prepared by the postoligomerization methodology developed by Harris et al. (20). A detailed description of the synthesis of the 11-mer oligonucleotides containing the (R)- and (S)-BDO and (R,R)- and (S,S)-BDE N2-guanines has been described previously (18). The 8-mer substrates containing the (R,R)- and (S,S)-intrastrand BDE N2-guanine-N2-guanine cross-links were synthesized as published previously (19).

To construct the templates for polymerase reactions, each adducted oligonucleotide was ligated by T4 DNA ligase (New England Biolabs Inc., Beverly, MA) with two flanking oligonucleotides in the presence of the complement 45-mer scaffold. The ligation products were purified via denaturing polyacrylamide gel electrophoresis. The sequences containing the BDO and BDE N2-guanine lesions are identical: 5'-AGAATGTGGAAGATACTGTGGGCAGGTGGTGAATGGTCTGGGCAATGTCGTTGACTGGGA-3', where the adducted G is underlined. The sequence containing the BDE N2-guanine-N2-guanine cross-link is as follows: 5'-CTAGAATGTGGAAGATACTGTGCATGGTCCAATGGTCTGGGCAATGTCGT-3', where the cross-linked guanines are underlined.

Oligodeoxynucleotides of anion-exchange grade purity were used as primers in the polymerase reactions and were obtained from the Midland Certified Reagent Co. (Midland, TX). Their sequences include 5'-ACGACATTGCCCAGACCATT-3', which is complementary to the BDO and BDE N2-guanine-adducted templates from positions -6 to -26 relative to the site of lesion. The same primer was used for the BDE N2-guanine-N2-guanine cross-link-containing substrates, being complementary from positions -4 to -24. 5'-ACATTGCCCAGACCATTGGA-3' was used as the -1 primer for the BDE N2-guanine-N2-guanine cross-link-containing substrates, and 5'-ATTGCCCAGACCATTCACCA-3' served as the -1 primer for the DNAs containing the BDO and BDE N2-guanine lesions. 5'-TTGCCCAGACCATTCACCAC-3' and 5'-GCCCAGACCATTCACCACC-3' served as the 0 and +1 primers, respectively, overlapping the lesion site in the BDO and BDE N2-guanine-adducted substrates.

Primer oligodeoxynucleotides were phosphorylated with T4 polynucleotide kinase (New England Biolabs Inc.) using [gamma -32P]ATP (PerkinElmer Life Sciences). The 32P-labeled primers were mixed with the oligonucleotide substrates in a molar ratio of 1:2 in the presence of 50 mM Tris-HCl (pH 7.0) and 100 mM NaCl, heated at 90 °C for 2 min, and slow cooled to room temperature. The completeness of the primer annealing was confirmed by electrophoresis through a native 7.5% polyacrylamide gel.

pol eta  Purification-- The glutathione S-transferase-pol eta  fusion protein was overexpressed and purified as described previously (4).

DNA Polymerase Reaction-- The pol eta  polymerase assays were carried out essentially as described by Johnson et al. (4). The reaction mixture (10 µl) contained 25 mM potassium phosphate buffer (pH 7.0), 5 mM MgCl2, 5 mM dithiothreitol, 100 µg/ml bovine serum albumin, 10% glycerol, 100 µM dNTPs (each of the four dNTPs or one, as indicated), 5 nM primer annealed to a template, and 2 nM glutathione S-transferase-pol eta . After incubation at room temperature for 20 min, reactions were terminated by the addition of a 10-fold excess loading buffer consisting of 95% (v/v) formamide, 20 mM EDTA, 0.02% (w/v) xylene cyanol, and 0.02% (w/v) bromphenol blue. The pol I (Klenow fragment) polymerase reactions were performed basically under the same conditions as the pol eta  reactions but in the presence of the buffer provided by the enzyme supplier (New England Biolabs Inc.). The reaction products were resolved through a 15% polyacrylamide gel containing M urea. Bands were visualized by autoradiography of the wet gels using Hyperfilm MP x-ray film (Amersham Pharmacia Biotech). Quantitative analyses of the results were performed using a PhosphorImager screen and ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA).

Steady State Kinetic Analysis-- Steady state kinetic assays were carried out under the same conditions as the DNA polymerase assays except that 1 nM pol eta  and 10 nM DNA substrates were used with various concentrations of one of the four nucleotides, and reactions were quenched after 5 min. DNA band intensities were quantitated using the PhosphorImager (Molecular Dynamics) and then used to calculate the rate of nucleotide incorporation as described previously (21). The rate of nucleotide incorporation was graphed as a function of nucleotide concentration, and kcat and Km parameters were obtained from the best fit of the data to the Michaelis-Menten equation.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Translesion DNA Synthesis by pol eta  on the (R)- and (S)-BDO and (R,R)- and (S,S)-BDE N2-Guanine-adducted DNA Substrates-- The structures of the BDO and BDE N2-guanine stereoisomers, which were examined in this study, are shown in Fig. 1. Among the butadiene epoxide guanine species that are formed as a result of the butadiene exposure, the N2-guanine adducts are relatively stable (16). In E. coli, replication efficiencies past the BDO and BDE N2-guanines are significantly reduced in vivo, and the presence of these lesions in DNA is a complete block to synthesis by E. coli pol I, II, and III in vitro (18).



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Fig. 1.   Structures of the BDO and BDE N2-guanines.

Primer extension reactions were carried out to test the ability of yeast pol eta  to perform translesion DNA synthesis on the BDO and BDE N2-guanine-adducted DNA substrates (Fig. 2A). Primers were designed that provided "running start" (-6 primer) and "standing start" (-1 primer) conditions. As shown in Fig. 2A, yeast pol eta  replicated through all four butadiene lesions, resulting in full-length products. However, pol eta  displays a strong stall site one nucleotide before the DNA lesion (lanes 3-6), suggesting an inhibition of nucleotide incorporation opposite the lesion. Interestingly, the bypass efficiency of pol eta  seems to show stereospecificity. On the BDO N2-guanine-containing substrates, as well as on the BDE N2-guanine adducts, translesion DNA synthesis was more efficient in the case of the S stereoisomers.



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Fig. 2.   DNA polymerase activity of S. cerevisiae pol eta  (A) and E. coli pol I (Klenow fragment) (B) under standing start and running start conditions on the monoepoxide- and diolepoxide-guanine-adducted templates. Each of the templates (ND = nondamaged, (R)-BDO = (R)-BDO N2-guanine, (S)-BDO = (S)-BDO N2-guanine, (R,R)-BDE = (R,R)-BDE N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine) was annealed to one of two primers. The DNA substrates (5 nM) were incubated for 20 min at 22 °C in the presence of all four dNTPs and S. cerevisiae pol eta  (2 nM) or E. coli pol I (Klenow fragment) (1 unit, as defined by New England Biolabs Inc.). Incubation of the nondamaged substrate under the same conditions but without polymerase was used as a negative control reaction. The positions of the 20-nt primers and the 51-nt (running start reaction) and 46-nt (standing start reaction) full-length products are indicated. *, position of the nondamaged G or the adducted G on the template.

Primer extension reactions using E. coli pol I (Klenow fragment) were also carried out on substrates containing the BDO and BDE N2-guanine adducts (Fig. 2B). These data confirm previous reports that BDO and BDE N2-guanines block DNA replication by pol I (18). This polymerase incorporated one nucleotide opposite the lesion but in contrast to the yeast pol eta , failed to extend the primer further on all four damaged substrates tested. Additionally, heterogeneity in the mobility of the final products (26-mer in the running start assays and 21-mer in the standing start assays) suggested nucleotide misincorporation in these reactions (lanes 3-6 and 9-12).

Next, the specificity of nucleotide incorporation by pol eta  opposite and downstream of these lesions was examined. To identify the nucleotide that was incorporated by pol eta  opposite the adducted base, single-nucleotide incorporation experiments were carried out using the -1 primer (Fig. 3). On a nondamaged substrate, pol eta  predominantly incorporated a C opposite G, but some T was also incorporated. In the case of the BDO and BDE N2-guanine-containing substrates, a C residue was the only base that was incorporated opposite the lesions. Taking into account the lower efficiency of primer extension by pol eta  on the adducted templates, the substrate to enzyme ratio in the reaction was changed from 5:2 to 1:4. Under these conditions, no nonextended primers were left in the reactions on all five substrates tested when dCTP was in the incubation mixture (data not shown). Again, in the presence of the dTTP, no primer extension was observed on any of the damaged DNA substrates, but primer extension occurred on the nondamaged template.



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Fig. 3.   Single-nucleotide incorporation by S. cerevisiae pol eta  on the monoepoxide- and diolepoxide-guanine-adducted templates using the -1 primer. Each of the templates (ND = nondamaged, (R)-BDO = (R)-BDO N2-guanine, (S)-BDO = (S)-BDO N2-guanine, (R,R)-BDE = (R,R)-BDE N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine) was annealed to the -1 primer. The DNA substrates (5 nM) were incubated for 20 min at 22 °C with each of the four dNTPs (--- = no nucleotide added, G = dGTP, A = dATP, T = dTTP, C = dCTP) and S. cerevisiae pol eta  (2 nM). The positions of the 20-nt primer and 22-nt products are indicated. *, position of the nondamaged G or the adducted G on the template strand.

Although in reactions with the -1 primer no significant level of misincorporation opposite the lesion was observed, the smearing of bands was noted one nucleotide beyond the lesion, particularly in the case of the (R)-BDO N2-guanine-adducted substrate (Fig. 2A). To determine whether this smearing was attributable to nucleotide misincorporation past the lesion site, single-nucleotide incorporation studies were performed on (R)-BDO N2-guanine-adducted template using a 0 primer, which contains a C opposite the damaged G. On this template, pol eta  extended 93% of the 0 primer with dCTP, 23% with dTTP, 6% with dATP, and 2% with dGTP (Fig. 4). A low level of primer extension was also observed in the presence of dTTP on the (S)-BDO N2-guanine-containing template. On the (R,R)- and (S,S)-BDE N2-guanine-containing templates, pol eta  incorporated only the C residue.



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Fig. 4.   Single-nucleotide incorporation by S. cerevisiae pol eta  on the monoepoxide- and diolepoxide-guanine-adducted templates using the 0 primer. Each of the templates (ND = nondamaged, (R)-BDO = (R)-BDO N2-guanine, (S)-BDO = (S)-BDO N2-guanine, (R,R)-BDE = (R,R)-BDE N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine) was annealed to the 0 primer. The DNA substrates (5 nM) were incubated for 20 min at 22 °C with each of the four dNTPs (--- = no nucleotide added, G = dGTP, A = dATP, T = dTTP, C = dCTP) and S. cerevisiae pol eta  (2 nM). The positions of the 20-nt primer and 21-nt products are indicated. *, position of the nondamaged G or the adducted G on the template strand.

To test whether any misincorporation occurred beyond one nucleotide downstream of the lesion site, we performed single-nucleotide incorporation experiments using a +1 primer (Fig. 5). No nucleotide misincorporation was observed on any of the damaged substrates examined, and pol eta  synthesized nearly the same amount of DNA on different damaged substrates when all four dNTPs were added to reactions.



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Fig. 5.   DNA polymerase activity of S. cerevisiae pol eta  under conditions of postlesion start on the monoepoxide- and diolepoxide-guanine-adducted templates. Each of the templates (ND = nondamaged, (R)-BDO = (R)-BDO N2-guanine, (S)-BDO = (S)-BDO N2-guanine, (R,R)-BDE = (R,R)-BDE N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine) was annealed to the +1 primer. The DNA substrates (5 nM) were incubated for 20 min at 22 °C in the presence of each of the four dNTPs or all four dNTPs (--- = no nucleotide added, G = dGTP, A = dATP, T = dTTP, C = dCTP, N4 = all four dNTPs) and S. cerevisiae pol eta  (2 nM). The positions of the 20-nt primer and the 44-nt full-length products are indicated. *, position of the nondamaged G or the adducted G on the template strand.

To quantitate the efficiency of pol eta -catalyzed synthesis past each of the BDO- and BDE-modified N2-guanines, steady state kinetic analyses were performed with both -1 and 0 primers. As shown in Table I, incorporation of dCTP opposite the (S)-BDO and (S,S)-BDE N2-guanines (-1 primer extension) was 200-300-fold less efficient than incorporation opposite the unmodified guanine, whereas incorporation opposite the R stereoisomers was 2000-3000-fold less efficient than incorporation opposite the unmodified guanine. The reduced efficiency for incorporating dCTP opposite BDO- and BDE-adducted N2-guanines is primarily a Km effect, not a kcat effect. Thus, there is a block to inserting dCTP opposite these lesions, as is also demonstrated by the pause site just prior to the adduct in Fig. 2A. The extent of the blockage depended on the stereochemistry of the adduct, and this result agrees with the data presented in Figs. 2A and 3. Interestingly, there is little block to extending from the C residue paired with the BDO- or BDE-modified N2-guanine (kinetics of the dCTP incorporation in reactions with 0 primer), as is also demonstrated by the lack of a pause site at the site of the adduct (Fig. 2A). In contrast to nucleotide incorporation opposite the lesion, no differences in efficiencies of elongation from the resulting base pair were observed. Thus, bypass efficiencies by pol eta  on the BDO- or BDE-modified N2-guanines are limited at the step of the nucleotide incorporation opposite the lesion but not at the extension step.


                              
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Table I
Efficiency of nucleotide incorporation by pol eta  on substrates containing the BDO and BDE N2-guanine

To further evaluate the accuracy of pol eta  replication through BDO and BDE N2-guanine adducts, kinetic analyses of nucleotide misinsertion were carried out, and frequencies of misincorporation were calculated as the ratio of kcat/Km of the incorrect nucleotide to the correct nucleotide (21). In reactions with the -1 primer, frequencies of misincorporation were below the limit of detection under conditions used for all four damaged substrates. Thus, pol eta  incorporates the correct nucleotide C quite accurately opposite N2-guanine modified with BDO or BDE. In experiments utilizing the 0 primer, high frequencies of misincorporation were observed in extension from C base-paired with the (R)-BDO N2-guanine. On this substrate, the frequencies of misincorporation were 2.0 × 10-2 for a T misincorporation and 6.2 × 10-4 for an A misincorporation. In all other cases, nucleotide misincorporation was below the limit of detection, which was approximately 5 × 10-4. Thus, kinetic data confirmed the results of the single-nucleotide incorporation experiment (Fig. 4), indicating that pol eta  is less accurate in extension from the base paired with (R)-BDO N2-guanine than with (S)-BDO N2-guanine.

Lack of Bypass of (R,R)- and (S,S)-BDE N2-Guanine-N2-Guanine Cross-links by pol eta -- Structures of the BDE N2-guanine-N2-guanine cross-links are shown in Fig. 6. Cross-linked adducts are believed to contribute to butadiene-mediated carcinogenesis (22, 23). Previously, in E. coli, both (R,R)- and (S,S)-BDE N2-guanine-N2-guanine cross-links were shown to be extremely inhibitory to replicative bypass in vivo, and E. coli DNA pol I, II, and III were shown to be completely blocked on the templates containing these cross-links in vitro (19). To examine whether yeast pol eta  can bypass these lesions, primer extension experiments were performed. As shown in Fig. 7, on the (R,R)- as well as on the (S,S)-BDE N2-guanine-N2-guanine cross-link-containing substrates, DNA synthesis by pol eta  was completely blocked just before the lesion, both under standing start and running start conditions.



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Fig. 6.   Structures of the BDE N2guanine-N2-guanine cross-links.



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Fig. 7.   DNA polymerase activity of S. cerevisiae pol eta  under standing start and running start conditions on the diolepoxide guanine-guanine cross-link adducted templates. Each of the templates (ND = nondamaged, (R,R)-CL = (R,R)-BDE N2-guanine-N2-guanine intrastrand cross-link-containing, (S,S)-CL = (S,S)-BDE N2-guanine-N2-guanine intrastrand cross-link-containing) was annealed to one of two primers (-4 or -1 primer). The DNA substrates (5 nM) were incubated for 20 min at 22 °C in the presence of all four dNTPs and S. cerevisiae pol eta  (2 nM). Incubation of the nondamaged substrate under the same conditions but without polymerase was used as a negative control reaction. The positions of the 20-nt primers and the 50-nt (running start reaction) and 47-nt (standing start reaction) full-length products are indicated. *, position of the nondamaged G or cross-linked G on the template.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Based on the ability of pol eta  to bypass a T-T dimer efficiently and accurately, it has been suggested that its active site is flexible enough to tolerate the distortion of the Watson-Crick geometry caused by the T-T dimer (6, 7, 24). However, such a flexibility of the polymerase active site should decrease its overall fidelity. Indeed, steady state kinetics assays of nucleotide incorporation have shown that pol eta  is a low fidelity enzyme. Both yeast and human pol eta  misincorporate nucleotides on undamaged DNA with frequencies of approximately 10-2-10-3 (6, 24, 25). Interestingly, the accuracy of replication by yeast as well as by the human pol eta  opposite a T-T dimer does not differ from that opposite nondamaged DNA (6, 7). The fact that both yeast (4, 26) and human (25) pol eta  do not possess any intrinsic proofreading exonuclease activity could explain in part the low fidelity of these polymerases. However, pol eta  has a lower fidelity than the other 3'right-arrow 5' exonuclease-deficient DNA polymerases (6, 24, 25), suggesting that its low fidelity derives from the relaxed requirement of its active site for correct base-pairing geometry. A flexible active site should enable pol eta  to bypass DNA lesions other than the T-T dimer. In agreement with this, both yeast and human pol eta  also bypass a 7,8-dihydro-8-oxoguanine lesion efficiently, and they do so by predominantly inserting a C opposite the lesion (11). In addition, both yeast (26) and human (27) pol eta  preferentially insert the correct nucleotide (C) opposite an N2-acetylaminofluorene-guanine. However, yeast pol eta  is unable to further extend DNA synthesis beyond the lesion (26). Human pol eta  can incorporate relatively efficiently one more nucleotide beyond the lesion, but only when the modified guanine is primed with a C (27).

Here it has been shown that yeast pol eta  can bypass (S)-BDO N2-guanine as well as (S,S)-BDE N2-guanine with 200-300-fold less efficient nucleotide insertion opposite the lesion relative to the nondamaged guanine. pol eta  can also bypass the (R)-BDO N2-guanine and (R,R)-BDE N2-guanine adducts, but these lesions pose an approximately 10-fold greater block to replication by pol eta  than their S stereoisomers. Thus, the efficiency of translesion DNA synthesis by yeast pol eta  is stereoisomer-specific. Blockage of the pol eta -catalyzed replication through the BDO and BDE N2-guanines occurs at the step of the nucleotide insertion opposite the lesion, not at the extension step. In its ability to effectively extend synthesis past the BDO and BDE N2-guanine adducts, yeast pol eta  differs from E. coli pol I, which fails to continue DNA synthesis beyond the lesion. Single-nucleotide incorporation experiments on BDO- and BDE N2-guanine-containing substrates and steady state kinetic data indicate that lesion bypass by pol eta  can be error-prone at the step of postlesion replication and that the accuracy of translesion DNA synthesis at this step can also be stereoisomer-specific. On three out of four substrates tested, namely on (S)-BDO, (R,R)-BDE, and (S,S)-BDE N2-guanine DNA adducts, nucleotide insertion opposite the lesion as well as elongation from the resulting base pair appeared to be quite accurate. On the (R)-BDO N2-guanine-containing substrate, pol eta  inserted the correct nucleotide opposite the lesion, but it showed a tendency for nucleotide misincorporation in elongation from the resulting base pair.

Stereoisomeric BDE N2-guanine-N2-guanine intrastrand cross-links were also examined in this study. However, these lesions were a complete block to synthesis by yeast pol eta , and in this case, synthesis terminated one base prior to the first adducted guanine. Interestingly, it has been recently demonstrated that human pol eta  is capable of inserting a C opposite the first G of a cisplatin-GG intrastrand cross-link, but incorporation of the second C was highly inefficient, even using higher concentrations of pol eta  in the reaction. When the cisplatin cross-link was primed with a CC opposite the lesion, bypass was achieved (27).

The N2-guanine adducts of stereoisomeric 1,3-butadiene metabolites are a complete block to synthesis by E. coli DNA polymerases I, II, and III. In contrast, yeast pol eta  can insert nucleotides opposite these lesions and is able to efficiently extend from the resulting base pair. The ability of yeast pol eta  to bypass N2-guanine butadiene adducts provides further support to the hypothesis (6, 7, 24) that in general, the pol eta  active site tolerates geometric distortions within DNA caused by these and other DNA-damaging agents. However, the inability of pol eta  to bypass an N2-guanine-N2-guanine intrastrand cross-link suggests that its active site is not flexible enough to adapt to the rather severe distortion imposed upon DNA by the cross-link.


    ACKNOWLEDGEMENTS

We thank T. M. Harris and C. M. Harris for modified DNAs, J. R. Carmical for template reagents, and NIEHS Center Molecular Biology Core for synthetic oligodeoxynucleotides.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants ES05355, S11-ES10018, and ES06676 (to R. S. L.) and GM 19261 (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 Holds the Mary Gibbs Jones Distinguished Chair in Environmental Toxicology from the Houston Endowment. To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 5.144 MRB, 301 University Blvd., Galveston, TX 77555-1071; Tel.: 409-772-2179; Fax: 409-772-1790; E-mail: rslloyd@utmb.edu.

Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M007867200


    ABBREVIATIONS

The abbreviations used are: pol eta , polymerase eta ; pol, polymerase; T-T, cis-syn cyclobutane pyrimidine; BDO, butadiene monoepoxide; BDE, butadiene diolepoxide; nt, nucleotide(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Johnson, R. E., Washington, M. T., Prakash, S., and Prakash, L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12224-12226[Free Full Text]
2. Woodgate, R. (1999) Genes Dev. 13, 2191-2195[Free Full Text]
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