422
Chem. Res. Toxicol. 1995, 8, 422-430
The Efficiency of Translesion Synthesis Past Single Styrene Oxide DNA Adducts In Vitro Is Polymerase-Specific Gary J. Latham,$>$,§ Constance M. Thomas M. and R. Stephen Lloyd*$§
and
Center in Molecular Toxicology and Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77550-1071 Received November 16, 1994@
In order to examine the effect of adenine N6 adducts of styrene oxide (SO) on DNA replication, 33-mer templates were constructed bearing site-specific and stereospecific SO modifications. Both R- and S-SO adducts were introduced a t four different base positions within a sequence containing codons 60-62 from the human N-rus gene. The resulting eight templates were replicated in primer extension assays using the Klenow fragment, Sequenase 2.0, T4 polymerase holoenzyme, polymerase a, and polymerase p. Replication of the damaged templates was analyzed under conditions defining single andlor multiple encounters between the polymerase and the substrate. Polymerization by all five enzymes was sensitive to both the local sequence context and the chirality of the SO adduct. For example, R-SO lesions placed a t the third position of N-ras codon 61 were readily bypassed, whereas stereochemically-identical lesions in other sequence contexts were often poor substrates for replication. Similarly, R- and S-SO adducts introduced within identical sequences were often bypassed nonequivalently. Significantly, the degree of adduct-directed termination and translesion synthesis during replication was also dependent on the choice of polymerase. Although SO adducts directed termination either opposite the lesion or 1 base 3’ t o the damage using all five polymerases, templates that were poor substrates for bypass synthesis with one enzyme were often read-through much more efficiently when a different polymerase was used. Thus, the activities of these enzymes on the SO-modified substrates produced replication profiles, or “fingerprints”, that were unique to each polymerase.
Introduction Styrene is a versatile building block in the development of resins and plastics and one of the most widely produced industrial organic chemicals (1).Those at risk for styrene exposure include industrial workers, during the development of styrene materials, and the general public, as a result of improperly disposed styrene products. The genotoxicity of styrene has been extensively investigated (for a review, see ref 21, with indications that the chemical is both a mutagen in prokaryotes and eukaryotes and a carcinogen in rodents. The detrimental genetic effects of styrene exposure have been primarily attributed to a metabolite, styrene oxide (SO),l that is generated during the cytochrome P450-dependent oxidation of styrene ( 3 ) . The reaction of SO with DNA is complicated by the fact that (i) the metabolism of styrene to SO creates a mixture of R and S isomers (41, (ii) SO can react at a number of nucleophilic sites in nucleosides, including guanine N-7, guanine N2, guanine 06,adenine N6, cytidine N4, and thymine C-3 (51,and (iii) SO adducts can arise from either a or p attack at the epoxide, depending on the site of modification (6, 7). Thus, the reaction of SO with
* To whom correspondence should be addressed. Tel: (409) 7722119; Fax: (409) 772-1790. Center in Molecular Toxicology, Vanderbilt University. Department of Biochemistry, Vanderbilt University. 5 University of Texas. ‘I Department of Chemistry, Vanderbilt University. @Abstractpublished in Advance ACS Abstracts, March 1, 1995. Abbreviations: SO, styrene oxide; KF, Klenow fragment; BSA, bovine serum albumin; DTT, dithiotheitol; gp43, gene product 43; gp441 62, gene products 44/62; gp45, gene product 45. 0893-228x/95/2708-0422$09.00/0
DNA creates a multiplicity of adducted structures, confounding the identification of specific adducts responsible for styrene-induced genotoxicity. The advent of chemical strategies to create a single DNA adduct within an oligodeoxynucleotide of defined sequence (8-10) has proven invaluable in linking the chemistry of adduction with the biological consequences of DNA damage. The biological consequences of any DNA lesion are a function of the size, site, and stereochemistry of the adduct, the sequence context of the DNA template, the nature of the polymerase replicating the damage, and the repair competence of the cell (11-21). In a previous study (161,we presented data supporting the hypothesis that adduct chirality and local sequence context modulate the replication fate of adenine N6-SO lesions within the human N-ras codon 61 sequence in a bacterial test system and in an in vitro replication assay. In this report, we expand the database of SO modifications to include R - and S-SO adducts (Figure 1)in four sequence contexts and analyze the contributions of the Klenow fragment (KF), Sequenase 2.0, bacteriophage T4 polymerase holoenzyme, polymerase a, and polymerase p in polymerizing past these adducts using primer extension assays. The results presented herein support earlier findings that replication by a specific polymerase is sensitive to both adduct stereochemistry and the local sequence context. Significantly, these data also demonstrate that the efficiency of synthesis past SO adducts is strongly dependent on the characteristics of the particular polymerase that copies the damaged template.
0 1995 American Chemical Society
In Vitro Replication of Styrene Oxide DNA Adducts
Q-C-CH20H HN
R-Styrene Oxide
SStyrene Oxide
Figure 1. Structures of the R and S stereoisomers of SO adducts on the adenine N6 position.
1 2 3 4 5 6 7 8 9 SO-adducted 11-mer+ Unmodified 11-mer+ Figure 2. Gel mobilities of 32P-labeledunmodified and SOmodified 11-mers. Lane 1, unmodified 11-mer; lane 2, R(6093)-S0 11-mer;lane 3, S(60t3)-S0 11-mer; lane 4, R(6112)-SO11-mer; lane 5,S(6192)-S011-mer; lane 6, R(61*3)-S0 11-mer; lane 7, S(61*3)-S0 11-mer; lane 8, R(62,2)-S0 11-mer; lane 9, S(62*2)-S0 11-mer.
Experimental Section Materials. Deoxynucleoside triphosphates (100 mM) were obtained from Pharmacia (Piscataway,NJ). KF (6 uniWpL) was purchased from Life Technologies, Inc. (Bethesda, MD), Sequenase 2.0 (13 units/pL) from U.S. Biochemical (Cleveland, OH), and human polymerase a (1.5 units/pL) from Molecular Biology Resources, Inc. (Milwaukee, WI).Rat polymerase /3 was a gift from Dr. S. H. Wilson, University of Texas Medical Branch. T4 polymerase holoenzyme (consisting of gene products 43,44/62, and 45) was kindly provided by the laboratory of Dr. B. M. Alberts, University of California a t San Francisco. Characterization of SO-Adducted11-mers. The starting materials for all in vitro studies were oligodeoxynucleotides comprised of an 11-base fragment derived from codons 60-62 of the human N-ras gene. These oligomers were synthesized by the method of Harris et al. (101,and purified as described (16). A total of eight SO-adducted 11-mers were prepared, containingR- or S-SOlesions at four different adenine positions within the human N-ras codon 60-62 sequence. The data presented in Figure 2 reveals the results of 32P-end-labeling analysis of the starting oligomers. As expected, the adducted 11-mers displayed a retarded mobility with respect to the unmodified 11-mer. Moreover, subtle differences in mobility were apparent between R- and S-SOoligomers adducted at the same position, as well as among chemically identical lesions in different sequence contexts. Preparationof SO-AdductedTemplates. The SO-modified 33-mers used as templates for primer extension assays were created by ligating purified SO-modified 11-mers to 22-mer oligodeoxynucleotides in the presence of a 27-mer scaffold as described (16). This construction created a 33-base product comprised of the adducted 11-mer at the 5’ region of the template and the 22-mer bridge oligomer at the 3’ end. The sequence of the 33-mer template is:
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 423 33-mer template (either SO-modified or unmodified) in the reaction buffer (55mM Tris.HC1, 11 mM MgC12, 55 mM KC1, and 5.5 mM /I-mercaptoethanol, pH 7.4 at 37 “C). Annealed template-primers were subjected to electrophoresisthrough 10% polyacrylamide native gels and quantified using the PhosphorImager (Molecular Dynamics); for each of the 9 templates, >90% of available primers were annealed. Nucleotides were then added to a final concentration of 100pM each and the reactions initiated with either limiting or excess enzyme in a volume of 20 pL to promote single or multiple encounters between the polymerase and the template-primer. Single “hit” conditions between enzyme and substrate were achieved by using a 1 : l O molar ratio of po1ymerase:template-primer and limiting the reaction time such that most primers were not extended. Multiple hit conditions were satisfied by using an excess of enzyme over substrate and long reaction times such that most primers were lengthened. The average number of hits was approximated by quantifying the level of primer signal at the unextended position in the gel and modeling the average number of hits with the Poisson distribution. Primer extension assays using the T4 polymerase holoenzyme employed the following reaction mixture (final concentration): 0.1 mg/mL bovine serum albumin (BSA), 1 mM dithiotheitol (DTT), 1.25 mM ATP, 0.3 M deoxynucleotide triphosphates (dNTPs) (each), 25 nM template-primer, 5 pg/mL gene product 43 (gp43), 24 pg/mL gp44/62, 7 pg/mL gp45,33 mM TrisOAc (pH 7.8), 66 mM KOAc, and 10 mM MgOAc. Reactions were initiated by adding 7 pL of annealed template-primer and dNTPs to 3 pL containing proteins 43, 44/62, and 45 and incubated for time periods ranging from 30 s to 45 min. Analyses of the replication profile of polymerase a using SOadduded templates employed a reaction mixture containing 0.14 mg/mL BSA, 1.4 mM DTT, 0.3 M dN”F’s (each), 25 nM templateprimer, 33 mM TrisOAc (pH 7.8),66 mM KOAc, 10 mM MgOAc, and either 0.3 unit (single hit conditions) or 1.5 units (multiple hit conditions) of polymerase. All reactions were terminated with stop buffer (95% formamide, 20 mM EDTA, 0.1% bromophenol blue, and xylene cyanole) and aliquots separated on 15%polyacryamide sequencing gels. Quantification of replication products was accomplished using the PhosphorImager (Molecular Dynamics). QuantitativeAnalyses. Quantitative comparisons among KF, Sequenase 2.0, and polymerase a were accomplished only after the basal activities of each enzyme on unmodified DNA were normalized according to the following formula:
normalized bypass efficiency = (fraction of molecules resulting from bypass synthesis on the damaged template)/(corresponding fraction of molecules resulting from synthesis on the control template) The calculated value is insensitive to loading error and independent of the average number of enzyme encounters per template-primer as long as single hit conditions are valid. Consequently, this correction reveals that if two polymerases synthesize the same number of product molecules upon bypass, the enzyme that extends primers more poorly on the control template (i.e., exhibits a lower activity) will have the higher bypass efficiency.
5’-CGG&C@G&4GAATTCGTCGTGACTGGGAAAAC-3’
Results
where the underlined bases represent sites bearing single adducts of R- or S-SO. Primer Extension Assays. Assay conditions and procedures used for studying replication by KF (large fragment of DNA polymerase I), Sequenase 2.0 (modified T7 polymerase), and polymerase B have been described (22). Briefly, 50 fmol of 17-merprimer (M13 universal primer) end-labeled with [y-32P]ATP a t the 5’ terminus was annealed with a 5-fold excess of
Rationale for In Vitro Replication with SO-Adducted Templates. In order to investigate the effects of in vitro replication upon DNA modified by SO, eight 11-mers bearing site-specific and stereospecific adenine N6 lesions of SO were used to construct 33-base oligodeoxynucleotide templates. For clarity, the adducted templates are distinguished by the nomenclature X-”p), where X = the enantiomer of SO (either R or S),y = the codon
424 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
60.3 61.2 61.3 62,2 wtRS R S R S R S
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Latham et al. 60.3 61,2 61.3 62.2 wtRS R S R S R S
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G soA
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soA
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Figure 3. Primer extension of SO-adductedtemplates by the Klenow fragment. (A) KF primer extension under single hit conditions. The sequence of the template strand is given to the left of the autoradiograph.The unmodified template is denoted “wt” (wild-type). “SO” marks the site-specifically adducted bases at either the third position of N-rus codon 60 (60,3),the second position of N-rus 61 (61,2),the third position of N-rus 61 (61,3),or the second position of N-rus 62 (62,2). “R” and “S”designate the chirality of the SO lesion at the modified site. Single hit conditions were accomplished by the addition of 5 fmol of enzyme (6 units/pL) in the presence of 50 fmol of template-primer. Reactions were incubated for 5 min. The average number of encounters between KF and the adducted templates ranged from 0.39 to 0.62. (B)KF primer extension under multiple hit conditions. Reactions were initiated with 500 fmol of enzyme and stopped after 60 min. The average number of encounters between KF and the adducted templates ranged from 2.2 to 3.5.
modified by the SO lesion (60, 61, or 62), and z = the base position (1,2, or 3) within that codon that contains the adduct. The damaged templates (and a control, unmodified template) were annealed to 32P-end-labeled 17-mer primers and replicated with various purified polymerases in primer extension assays. Replication was performed under conditions satisfylng single and/or multiple encounters between the polymerase and the template-primer. The defined nature of the adducted substrates, coupled with the ability to conduct replication in a controlled in vitro environment, permitted a systematic study of the contributions of SO chirality, sequence context, and the replicating polymerase to the effect of specific lesions during DNA synthesis. Replication by the Klenow Fragment. KF (large fragment of DNA polymerase I from Escherichia coli) is a 68 kDa protein with both polymerase and 3’-5’ exonuclease activities (23). The results of KF primer extension on the SO-modified 33-mers are shown in Figure 3. Consistent with other literature reports of polymerase action on bulky DNA adducts (24-261, SO lesions inhibited replication by blocking primer extension either opposite the adduct or 1 base 3’ to the damage (Figure 3A). Furthermore, the replication profile for each damaged template under single hit conditions was dependent on the stereochemistry of the adduct. For example, polymerization on the R(6193)-S0 template created levels of fully extended (33-base) primers comparable to the unmodified (wt) template, a result consistent with previous work (16). However, the S(6193)-S0 33-mer, which differs from the R(6193)-S0 template only in the chirality of the adduct, was a very poor substrate for lesion bypass synthesis. Analyses of the replication data in which sequence context effects were measured re-
vealed major differences in the severity of lesion-induced blockage such that bypass of the R(6193)-S0 lesion was very efficient, whereas bypass of the other 3 R-SO adducts (i.e., R(6093)-S0, R(61*2)-S0, and R(62y2)-SO) was extremely limited. In general, product quantification revealed that KF fully extended primers (to a 33-base product) on R-SO-containingtemplates more completely than primers on S-SO-containingtemplates (i.e., full length extension of primers on R(6192)-S0was more efficient than S(61,2)-SO,R(61,3)_SO> S(613)-SO,and R(6229-SO > S(62,2)_SO). In a previous study (16), we reported that the primer extension on R /S(61,2)-S0 and R /S(61y3)-S0 templates by equimolar amounts of KF was poorest for R(61*2)-SO; however, this finding was not substantiated by the data in Figure 3. Upon further investigation, the difference in replication between R(61*2)-S0 and R(61y3W0was attributed to an underestimation in the concentration of the R(61*2)-S0 template in the earlier report, causing incomplete annealing with the labeled primer. In actuality, under single hit conditions, the R(61*2)-S0 template was second only to R(6193)-S0 in the level of fblly extended primers produced by KF. Multiple encounters between the KF and the templateprimer permitted the extension of prematurely truncated replication products to full length. As illustrated in Figure 3B, templates that were poor substrates for replication under single hit conditions (e.g., R / S(6292)-SO) could be completely extended after repeated catalytic cycling, although stop sites in the vicinity of the adduct remained (albeit of lesser intensity). Some intermediate length products were more readily extended than others during repeated encounters between enzyme and substrate (Figure 3A,B, compare stop sites 1base 3’ to the adduct in the R(62*2)-S0 and S(6292)-S0 lanes). Interest-
In Vitro Replication of Styrene Oxide DNA Adducts 60.3 61.2 61.3 62.2 wtRS R S R S R S
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Chem. Res. Toxicol., Vol. 8, No. 3, 1995 425 60.3 61.2 61.3 62.2 n n n n wtRS R S R S R S
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Figure 4. Primer extension of SO-adducted templates by Sequenase 2.0. (A) Sequenase 2.0 primer extension under single hit conditions (reprinted with permission of the American Society for Biochemistry and Molecular Biology, Inc., Bethesda, MD). Single hit conditions were accomplished by the addition of -5 fmol of enzyme (13units/pL) in the presence of 50 fmol of template-primer. Reactions were incubated for 30 min. The average number of encounters between Sequenase 2.0 and the adducted templates ranged from 0.17 to 0.36.Panel A has been published elsewhere (22), but is shown here (with permission) for convenient comparison with panel B. (B) Sequenase 2.0 primer extension under multiple hit conditions. Reactions were initiated with 1000 fmol of enzyme and stopped after 90 min. The average number of encounters between Sequenase 2.0 and the adducted templates ranged from 1.0 to 2.0.
ingly, translesion synthesis of the R / S(6093)-S0 templates was extremely inefficient even in the presence of excess enzyme and long reaction times. Replication by Sequenase 2.0. The replicative processing of SO-containingtemplates by Sequenase 2.0 (an exonuclease-deficient form of bacteriophage T7 polymerase) is shown in Figure 4. Although the replication profile of Sequenase 2.0 is similar to that of KF, there are significant differences. Unlike KF, Sequenase 2.0 replicated S(6193)-S0 and S(62*2)-S0 templates to form substantial amounts of h l l length products (Figure 4A).2 Indeed, Sequenase catalyzed 6-fold and 11-fold more 33base product molecules on the S(6193)-S0 and S(62p2)-S0 templates, respectively, than KF (when corrected to an equal number of hits by both enzymes) even though the overall activity of Sequenase 2.0 in replicating the control template was poorer than KF in this assay (see below). Moreover, Sequenase fully extended primers on S-SOcontaining templates more completely than primers on R-SO-containing templates (i.e., full length extension of primers on S(6093)-S0 templates was more efficient than R(6093)-SOtemplates, S(6G')-SO> R(61,2)-SO,and S(623-SO > R(62*2)-SO), whereas the opposite was true for KF (see below and Table 1)).Thus, the replication fate of SOmodified 33-mers was determined by the choice of polymerase. Similar to the replication profile of KF, repeated encounters between Sequenase and the damaged substrates promoted the extension of many partially replicated products to completion (Figure 4B). In addition, Sequenasecatalyzed the extension of a greater proportion of product molecules to a position opposite the lesion relative to those products extended to 1 base prior to Although Figure 4A has been previously published (ZZ),we include these data so that comparisons with the unpublished data of Figure 4B are convenient.
reaching the adduct (Figures 3B and 4B, compare R(62%30 lanes). Sequenase 2.0 was also able to extend primers to R /S(60>3)-S0 templates to completion with much greater efficiency than KF. Lastly, Sequenase 2.0 catalyzed a nontemplated 1base blunt-end addition to many primers, creating a 34-base product. End addition often occurs at significant levels when template-primers are extended by an excess of certain exonuclease-deficient polymerases (27). Replicationby Human Polymerase a. Polymerase a is a multiprotein complex composed of both polymerase and primase activities (28). These functions, in combination with the low processivity of the enzyme, ideally suit polymerase a for lagging strand synthesis (i.e., initiator DNA synthesis) (29). Primer extension studies were undertaken with polymerase a to evaluate adduct-specific replication by a eukarS;otic enzyme that participates in chromosomal synthesis. As illustrated in Figure 5A, polymerase a was less processive than KF or Sequenase 2.0, and more sensitive to SO damage in the templateprimer stem, as evidenced by substantial termination 1 base 5' to the adduct. Furthermore, polymerase a displayed roughly comparable bypass efficiencies for SO lesions in all templates, except the R(6193)-S033-mer. Consistent with results of KF and Sequenase 2.0 replication, R(61y3)-S0 was the most facile substrate for translesion synthesis by polymerase a. However, the S(6173)-S0 template promoted the second most fully extended primer upon incubation with polymerase a, contrary to the results obtained with KF and Sequenase 2.0. As expected, all eight SO adducts were bypassed to create significant levels of fully extended primers under multiple hit conditions (Figure 5B). Replication by Rat Polymerase /?.Polymerase p is a 39 kDa enzyme implicated in DNA repair synthesis in eukaryotes (30).When standard template-primers are
426 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Latham et al.
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Figure 5. Primer extension of SO-adductedtemplates by human polymerase a.(A) Polymerase a primer extension under single hit conditions. Single hit conditions were accomplished by the addition of -15 fmol of enzyme (1.5 units/pL) in the presence of 250 fmol of template-primer. Reactions were incubated for 10 min. The average number of encounters between polymerase a and the adducted templates ranged from 0.45 to 0.87. (B) Polymerase a primer extension under multiple hit conditions. Reactions were initiated with -75 fmol of enzyme and stopped after 75 min. The average number of encounters between polymerase a and the adducted templates ranged from 4.4 to 4.6. 60,3 61,2 61,3 62,2
n n n n
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Prime Figure 6. Primer extension of SO-adducted templates by polymerase p. Reactions were initiated by the addition of 6000 fmol of enzyme in the presence of 50 fmol of template-primer and stopped after 30 min.
used as substrates, polymerase p replication is distributive; however, when presented with a short, gapped DNA substrate, the mode of synthesis shifts to a processive one (31). Due to the proposed role of polymerase p in DNA repair, we were interested in testing the activity of
this protein on DNA damaged by SO. Under the conditions of our assay, polymerase p synthesis was distributive; as a result, single hit polymerization assays were not meaningful during a single encounter due to the inability of the polymerase to replicate up to the damaged base. Consequently, Figure 6 presents the results of primer extension with polymerase p under multiple hit conditions. In contrast to the action of the three-polymerases discussed above, the poorest substrates for full extension using polymerase 16 were the R / S(6192)-S0 templates. This is surprising when one considers that even those templates bearing SO lesions very close to the 5' end (i.e., R /S(6093)-SO) were more efficiently replicated than the R /S(61y2)-S0 33-mers. Thus, again, these results demonstrate that translesion synthesis was not only polymerase-specific, but also sensitive to the adduct sequence context. Replication by Bacteriophage T4 Polymerase Holoenzyme. Bacteriophage T4 replicates its DNA using a complex of proteins referred to as the T4 polymerase holoenzyme (32). These proteins include gp43 (polymerase), gp44/62 (brace proteins), and gp45 (sliding clamp), although the functional definition of the holoenzyme has been recently expanded to include gp32 (single-strandedbinding protein) (33). Moreover, the T4 polymerase assembly is an excellent model for the leading strand replicases of both E. coli and eukaryotic organisms. In order to test the effects of defined DNA damage on the activity of a true replicase involved in genomic DNA synthesis, we examined the replication characteristics of the T4 polymerase holoenzyme on SO-modified 33-mers. Under the reaction conditions employed, only multiple hit conditions could be satisfied (Figure 7). However, to underscore any differences in adduct bypass, the kinetics of synthesis by T4 polymerase were analyzed after 30 s and 9 min of replication (Figure 7, panels A
In Vitro Replication of Styrene Oxide DNA Adducts
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 427
60-3 61,2 61,3 62-2 wtRSRS R S R S
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Figure 7. Primer extension of SO-adducted templates by T4 polymerase holoenzyme. (A) 30 s extension reaction with T4 holoenzyme. See Experimental Section for reaction conditions. (B) 9 min extension reaction with T4 holoenzyme. At both 30 s and 9 min time points, >98.5% of available primers were extended on SO-damaged templates.
Table 1. Normalized Bypass Efficiencies of KF, Sequenase 2.0, and Polymerase a Past Single SO DNA Adducts in Different Seauence Contextsa
KF Sequenase 2.0 a
1.0 1.0 1.0
0.057 0.22 0.48
0.044 0.44 0.37
0.38 0.65 0.45
0.11 0.68 0.36
0.86 0.96 0.85
0.11 0.52 0.25
0.24 0.27 0.41
0.14 0.65 0.22
a Normalized values were calculated by dividing the fraction of product molecules resulting from translesion synthesis on a given damaged template by the corresponding fraction computed from replication on the unmodified template. Thus, larger values correlate to more efficient bypass of the adducted base. The data presented were obtained from PhosphorImager analysis of the products from the single hit assays with each of the respective polymerases.
and B, respectively). Significantly, the holoenzyme replicated past SO lesions with greater facility as they were positioned increasingly further from the 5' terminus of the template after 30 s of synthesis (Figure 7A). Polymerization on the R(62*2)-S0 template, however, was not consistent with this observation as the R(62*2)-S0 lesion presented a very strong block to replication 1base 3' to the adducted base, thereby limiting bypass synthesis. Nevertheless, when the reaction time was extended to 9 min, bypass of the R(62*2)-S0 lesion was dramatically enhanced, based on the level of full length primer (Figure 7B). In contrast, the change in the level of completely replicated primers extended on the R(61$2)-S0 template from 30 s to 9 min was relatively minor even though the number of 111 length primers was comparable for R(612)-S0 and R(62,2)-S0 after 30 s of synthesis. Thus, Figure 7 indicates that bypass of adducts in different sequence contexts can be achieved by kinetically distinct mechanisms. Quantitative Analyses of Polymerase Bypass Efficiencies. A rigorous, quantitative comparison of the efficiencies of translesion synthesis displayed by the polymerases examined in this study was restricted to those enzymes analyzed under single hit ~onditions.~ One factor that must be considered for these comparisons to be valid is whether the enhanced bypass of DNA damage
by one polymerase relative to another is due to an intrinsic ability of the polymerase to replicate past the lesion (reflecting unique structure-function relationships of the enzyme) and/or merely a greater specific activity of the enzyme for overall polymerization. Thus, comparisons of the bypass efficiencies by different polymerases are most meaningful when the proteins are normalized for activity on the same unmodified DNA. In this way, an increase in read-through by one polymerase over another can be attributed to an inherent proclivity of the enzyme to bypass adducted sites rather than a higher enzyme activity. As a result, we have normalized the efficiency of bypass of an adducted site by a given enzyme to the activity of that enzyme on the undamaged template. The results of this correction are displayed in Table 1. These data confirm the qualitative comparisons described above; that is, the efficiency of bypass is strongly dependent on the polymerase employed. For example, Sequenase 2.0 creates more fully extended primers than KF' on several of the SO-adducted templates even before normalization. Since Sequenase 2.0 has a lower activity on the control template than KF,4 the 3 Since the extent that intermediate length products are further extended by repetitive encounters is unknown in the multiple hit experiments, quantitative comparisons of product synthesis under these conditions are not valid.
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facility that Sequenase 2.0 synthesizes past SO is magnified after correction, resulting in a much greater bypass efficiency. In general, the data presented in Table 1 demonstrate that both Sequenase 2.0 and polymerase a bypass the SO lesions with higher efficiencies than KF. Moreover, the two SO-damaged templates that are readthrough by Sequenase with greater than 50% relative efficiency (e.g., S(61’3’ and S(62b2)) are 2-&fold more poorly bypassed by polymerase a and KF. Similarly, polymerase a replicates past the R(6033j and R(62,2) lesions significantly better than either KF or Sequenase 2.0. In short, quantitative analysis of the results of primer extension with these three polymerases demonstrates that the efficiency of translesion synthesis is dependent on the sequence context of the SO damage and the choice of polymerase.
Discussion The purpose of this study was to examine the in vitro replication consequences of site-specific and stereospecific adenine N6 adducts of SO by prokaryotic and eukaryotic polymerases. In addition, the hypothesis was tested that adduct chirality and sequence context modulate the ability of polymerases to bypass a specific lesion. To provide insight into this question, eight SO-adducted templates containing either R- or S-SO modifications in four different sequences were synthesized. We report here that the efficiency of replication past the styryladducted templates varied significantly depending on the choice of polymerase. Furthermore, we find that stereochemically-identical SO adducts in different sequences were often replicated dissimilarly, as were enantiomeric SO adducts in homologous sequence contexts. In order to evaluate the role of the polymerase in replicating site-defined SO adducts, conditions were designed to promote an average of single and/or multiple cycles of catalysis. As a result, the effect of polymerase concentration on translesion bypass could be assayed at a precise, quantitative level. Historically, DNA adducts are known to have two fates during replication: (i) blockage of the polymerase apparatus either opposite or 1base 3’ to the damage, and/or (ii) translesion synthesis, promoting full length replication of the template. Recently, a third fate of adduct replication, namely, the termination of synthesis a t a defined distance after translesion replication but prior to complete primer extension, has been reported with HIV-1 reverse transcriptase (22). In contrast, the replication profiles of KF, Sequenase 2.0, T4 polymerase holoenzyme, polymerase a, and polymerase /3 documented in this study were consistent with the premature termination of synthesis in the immediate vicinity of the lesion. The development of stop sites 1 base 3‘ to the adduct indicates that nucleotide insertion opposite a styryl-modified adenine was not catalytically favorable. Moreover, when the extension of these primers was templated by the damaged base, the resultant primers, in turn, were often poorly extended, as evidenced by strong stop sites opposite the adducted base. Replication by Sequenase 2.0 and polymerase a also revealed significant termination 1base 5’ to the lesion (Figures 4 and 5), indicating that the adduct moiety can perturb catalysis even when the damage is positioned in the template-primer stem. The lower activity of Sequenase in relation to K F in the single hit assays reflects a lower processivity andor slower rate of nucleotide insertiodextension during a single binding event.
Latham et al. However, it is not clear from our data whether the termination sites observed represent sites of dissociation of the polymerase from the template-primer or continued polymerase binding and a slower nucleotide insertion rate. We have conducted preliminary studies to determine which bases are incorporated opposite the SOadducted base in the event of bypass and note that the polymerase tested, HIV-1reverse transcriptase, inserted nucleotides opposite dA-SO in the rank order dTTP >> dCTP > dATP dGTP a t both R(61,2)and S(61,2)-S0 basesS5 Whether or not the poor kinetics of bypass synthesis are a consequence of nucleotide misincorporation at sites other than R/S(61z2) remains to be seen. Indeed, Fry and Loeb (34)have reported that a natural pause site for polymerase a is a hotspot for nucleotide misinsertion. A number of groups have examined the action of different polymerases in the replication of site-specific adducts (18, 24, 25, 35, 36). For example, BelguiseValladier et al. (24) analyzed the role of KF, Sequenase 2.0, and polymerase I11 holoenzyme in the bypass of single adducts of N-2-(acetylamino)fluorene and cisdiamminedichloroplatinum(II), with the finding that the extent of primer extension depended on which polymerase was employed. However, very little bypass of the adducts was observed in this study, precluding a more exhaustive comparison of polymerase activities. The SOcontaining templates investigated in this work revealed considerable heterogeneity in replicative capacities, ranging from a complete block (e.g., R(60!3)-S0 replication by KF and T4 polymerase holoenzyme) to facile bypass (e.g., R(61,3)-S0 replication by all of the polymerases tested) under multiple hit conditions. Thus, the spectrum of bypass efficiencies observed with styryl-modified templates enabled the performance of various polymerases to be assessed over a large dynamic range. Examination of the data lends insight into structurefunction relationships during the replication of damaged DNA. For example, the R(6133)-S0 adduct was readily bypassed by all the polymerases assayed, indicating that this lesion exhibits structural features that each polymerase recognizes and responds to in a fundamental, functionally homologous manner. In contrast, translesion synthesis of the S(61,2)-S0 modification under single hit conditions was very poor for KF, moderately efficient for polymerase a,and efficient for Sequenase 2.0 (Table 1). Thus, the structure of the S(61,2j-S0 modification during catalysis had very different effects on the three polymerases. The implication, then, is that catalytic interpretation of the damaged substrate structure relies on multiple influences, including adduct chirality and sequence context. Furthermore, comparisons of the duplex structure of the R(6133)-S0 and R(61W3011-mers, which have been resolved by NMR spectroscopy,6 do not reveal any prominent structural differences in the two adducted oligomers. Yet, the replication profiles of these two substrates differ dramatically. Thus, either the replication apparatus is sensitive to very minor structural differences in the template-primer or, more likely, the duplex structure in solution does not adequately reflect the structure of the adducted template-primer when bound to a catalytically competent enzyme. The eight adducted templates analyzed by primer extension contained SO lesions in four different 3-base G. J. Latham and R. S. Lloyd, unpublished observations. B. Feng and M. P. Stone, personal communication.
I n Vitro Replication of Styrene Oxide D N A Adducts sequence contexts: 5’-GAC-3’ (R/S(60p3j-S0template), 5‘CAA-3‘ (R/S(61,2j-SO), 5’-AAG-3’ (R/S(61z3j-SO),and 5’G u - 3 ‘ (R/ S(62r2j-SO), where the underlined base marks the adducted site. Definitive comparisons among these sequence contexts are complicated by the possibility that, under single hit conditions, adducts located very close to the 5’ end of the template (e.g., RIS(60z3j-SO)may be poorly replicated due to destabilizing end effects (see below). However, comparisons of adducts positioned more internally in the template are instructional. For instance, the local sequence contexts of R(6133)-S0 (5’-AAG3’) and R(6212j-S0(5’-GAA-3’) both have 5’ and 3‘ purines flanking the damage, $t the efficiency of replication past these sites was very different; whereas the R(61,3)-S0 lesion was bypassed more readily than any other adduct, pausing at the R(62s2j-S0modification was severe even under multiple hit conditions. Thus, relatively conservative changes in sequence context can effect significant changes in the effkiency of translesion synthesis. The effect of an exonucleolytic, editing activity during the replication of adducted DNA is known to increase fidelity and promote catalytic idling at the damaged base (24,371. The impact of a polymerase editing complex was also evidenced during the synthesis of SO-adducted templates. A comparison of the replication profiles of each polymerase on R(6083)-S0 templates under multiple hit conditions demonstrated that complete primer extension only occurred when polymerases devoid of 3’-5’ exonucleolytic activity were assayed (i.e., Sequenase 2.0, polymerase a,and polymerase p). To test this hypothesis further, experiments were also conducted with a KF mutant that was 3’-5’ exonuclease-deficient. Unlike KF, which did not extend primers to R(60s3j-S0to full length (Figure 3B), the mutant enzyme bypassed the R(60,3)-S0 lesion efficiently to generate completely replicated products (data not shown). In addition, HIV-1 reverse transcriptase catalyzed the complete extension of most primers to the R(60,3j-S0 template (221, a consistent result since reverse transcriptase lacks an editing domain. One explanation for this finding is that exonucleolytic polymerases such as T4 holoenzyme and KF engage in nonproductive cycling a t the R(60,3)-S0damaged base, resulting from competition between nucleotide insertion and hydrolytic removal of the incorporated base. The inefficiency of this process may be exacerbated by the presence of a destabilizing adduct so closely positioned to the 5‘ terminus of the template, thereby accounting for differences in the replicative capacity of R(60,3j-S0 compared to the other 3 templates containing R-SO adducts. Thus, some polymerase-specific differences observed during replication were the consequence of dual polymerase-exonuclease activities. In conclusion, this paper demonstrates that the in vitro replication of SO-adducted templates is enantiospecific, sequence context dependent, and polymerase-specific. The complex interaction of these factors in determining adduct fate necessitates the development of a database describing how these factors influence one another. Only then can one hope to formulate general rules, if such rules exist, for understanding the molecular details of how the consequences of DNA adduction are manifested in cellular systems.
Acknowledgment. We gratefully acknowledge P. Horton for the synthesis and purification of the SOadducted oligomers, Dr. S. H. Wilson for providing rat polymerase p, and the laboratory of Dr. B. M. Alberts
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 429 for the gift of T4 polymerase holoenzyme. We also thank Drs. S. H. Wilson, W. A. Beard, R. Schrock, and K. A. Latham for helpful discussions during the course of this study. This work was supported by USPHS Grants ES00267, ES05355, ES05509, and ES07028, and ACS FRA381. References Bond, J. A. (1989) Review of the toxicology of styrene. CRC Crit. Rev. Toxicol. 19, 227-249. Barale, R. (1991) The genetic toxicology of styrene and styrene oxide. Mutat. Res. 257, 107-126. WHO (1983) Environmental Health Criteria 26: Styrene, World Health Organization, Geneva, Switzerland. Foureman, G. L., Harris, C., Guengerich, F. P., and Bend, J. R. (1989) Stereoselectivity of styrene oxide oxidation in microsomes and in purified P450 enzymes from rat liver. J. Pharmacol. Exp. Ther. 248,492-497. Savela, K., Hesso, A., and Hemminki, K. (1986) Characterization of reaction products between styrene oxide and deoxynucleosides and DNA. Chem.-Biol. Interact. 60, 235-246. Hemminki, K.,Forsti, A., Mustonen, R., and Savela, K. (1986) DNA adducts in experimental cancer research. J . Cancer Res. Clin. Oncol. 112, 181-188. Latif, F., Moschel, R. C., Hemminki, K., and Dipple, A. (1988) Styrene oxide as a stereochemical probe for the mechanism of aralkylation at different sites on guanosine. Chem. Res. Toxicol. 1, 364-369. Basu, A. K.,and Essigmann, J. M. (1988)Site-specifically modified oligodeoxynucleotides as probes for the structural and biological effects of DNA damaging agents. Chem. Res. Toxicol. 1, 1-18. Singer, B.,and Essigmann, J. M. (1991) Site-specific mutagenesis: retrospective and prospective. Carcinogenesis 12,949-955. Harris, C. M., L. Zhou, L., Strand, E. A., and Harris, T. M. (1991) New strategy for the synthesis of oligodeoxynucleotides bearing adducts at exocyclic amino sites of purine nucleosides. J . Am. Chem. SOC.113, 4328-4329. Basu, A. K.,and Essigmann, J. M. (1990) Site-specifically alkylated oligodeoxynucleotides: Probes for mutagenesis, DNA repair, and the structural effects of DNA damage. Mutat. Res. 233, 189-201. Bishop, R. E., and Moschel, R. C. (1991) Positional effects on the structure and stability of abbreviated H-ras DNA sequences containing 06-methylguanine residues at codon 12. Chem. Res. Toxicol. 4,647-654. Topal, M. D. (1988) 06-methylguanine mutation and repair is nonuniform. Carcinogenesis 9, 691-696. Horsfall, M. J.,Gordon, A. J. E., Burns, P. A,, Zielenska, M., van der W e t , G. M. E., and Glickman, B. W. (1990) Mutational specificity of alkylating agents and the influence of DNA repair. Enuiron. Mol. Mutagen. 15, 107-122. Burnouf, D.,Koehl, P., and Fuchs, R. P. P. (1989) Single adduct mutagenesis: Strong effect of the position of a single acetylaminofluorene adduct within a mutation hot suot. Proc. Natl. Acad. Sci. U.S.A. 86, 4147-4151. (16) Latham, G. J.,Zhou, L., Harris, C. M., Harris, T. M., and Lloyd, R. S. (1993)The replication fate of R- and S-styrene oxide adducts on adenine N6 is dependent on both the chirality of the lesion and the local sequence context. J. Biol. Chem. 268,23427-23434. (17) Singer, B., Chavez, F., Goodman, M. F., Essigmann, J. M., and Dosanjh, M. K. 11989)Effect of 3’ flanking neighbors on kinetics of pairing of dCTP or dTTP opposite 06-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used. Proc. Natl. Acad. Sci. U.S.A. 86, 8271-8274. (18) Shibutani, S., Takeshita, M., and Grollman, A. P. (1991)Insertion of specific bases during DNA synthesis past the oxidationdamaged base 8-oxo-dG. Nature 349, 431-434. (19) Fuchs, R. P. P. (1983) DNA binding spectrum of the carcinogen N-acetoxy-N-2-acetylaminofluorene significantly differs from the mutation spectrum. J . Mol. Biol. 177, 173-180. (20) Rodriguez, H., and Loechler, E. L. (1993) Mutational specificity of the (+I-anti-diol epoxide of benzo[alpyrene in a supF gene of an Escherichia coli plasmid: DNA sequence context influences hotspots, mutagenic specificity, and the extent of SOS enhancement of mutagenesis. Carcinogenesis 14, 373-383. (21) Randall, S. K.,Eritja, R., Kaplan, B. E., Petruska, J., and Goodman, M. F. (1987) Nucleotide insertion kinetics opposite abasic lesions in DNA. J . Biol. Chem. 262, 6864-6870. (22) Latham, G. J., and Lloyd, R. 5. (1994) Deoxynucleotide polymerization by HIV-1 reverse transcriptase is terminated by sitespecific styrene oxide adducts after translesion synthesis. J.Biol. Chem. 269, 28527-28530.
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