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Pawe Jaoszyski^1,a,*, Eiji Ohashi², Haruo Ohmori² and Susumu Nishimura¹

¹ Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
² Institute for Virus Research, Kyoto University, Shogoin-Kawaracho 53, Sakyo-ku, Kyoto, 606-8507, Japan

	Abstract

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Using fragments of human c-Ha-ras and mouse Ha-ras1 genes containing 8-hydroxyguanine (8-OH-G) in hypermutagenic codon 12, we analyzed the kinetics of DNA synthesis catalyzed by human Pol. This translesion DNA polymerase, belonging to the Y-family, was found to be moderately inhibited by the presence of 8-OH-G on either mouse or human templates. From our previous results, inhibition of various polymerases by 8-OH-G increases in the following order: Pol < Pol < Polß < Pol, showing that major replicative and repair polymerases are more sensitive to this lesion than enzymes belonging to the Y-family. In the direct mutagenesis experiments, Pol was found to be more mutagenic than Pol studied previously: it inserted dAMP more efficiently than dCMP opposite 8-OH-G. Pol was also able to cause indirect mispair (‘action-at-a-distance’ mutagenesis), this effect being more distinct on mouse templates. Two adjacent 8-OH-G residues in codon 12 inhibited Pol moderately and induced misincorporation of dAMP. However, this effect was not comparable to the strong relaxation of the enzyme specificity, observed previously in the case of Pol. Pol catalyzed incorporation (and misincorporation of dAMP) much more efficiently on mouse templates, human DNA fragments being distinctly worse substrates. Interestingly, in direct mutagenesis systems, the preference for dAMP over dCMP was nearly the same on mouse and human templates.

	Introduction

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Recent studies have provided much interesting data on particular roles of non-replicative DNA polymerases in mutagenic events. A group of minor polymerases was found to have the unique ability of translesion DNA synthesis, which may have essential significance for cell survival, mutagenicity and carcinogenesis (reviewed by Prakash & Prakash 2002). Enzymes of particular interest belong to the Y-family of DNA polymerases (Ohmori et al. 2001). This group includes polymerases , and , which were shown to be required for bypass of various forms of DNA damage (Burgers et al. 2001). Polymerase bypasses thymine-thymine cyclobutane dimers and thymine-thymine 6-4 photoproducts with low efficiency (Johnson et al. 2000a; Tissier et al. 2000), and benzo[a]pyrene as well as benzo[c]phenanthrene diol epoxides adducted to adenine (Frank et al. 2002). However, this enzyme is strongly blocked by the same diol epoxides adducted to guanine (Frank et al. 2002). Polymerase (Pol), encoded by the human Xeroderma pigmentosum variant (XPV) gene (Johnson et al. 1999; Masutani et al. 1999), was found to catalyze efficient and accurate replication past thymine dimers (Johnson et al. 2000b), and to play an important role in the error-free bypass of UV lesions in an in vivo yeast system (Yu et al. 2001). Moreover, Pol bypasses platinum complexes-DNA adducts efficiently, but in an error-prone manner (Veisman et al. 2000), and also 1,N⁶-ethenoadenine (Levine et al. 2001). Inaccurate replication by Pol was also shown in the case of abasic sites and benzo[a]pyrene adducts (Zhang et al. 2000a). Pol has also been examined in several experimental systems containing 8-hydroxyguanine (8-OH-G) [also known as 7,8-dihydro-8-oxoguanine, 8-oxo-G]. In the same year, two research groups reported efficient and accurate replication past this lesion (Haracska et al. 2000) and misinsertion (Zhang et al. 2000a). Recently, we investigated the mutagenicity of this and other enzymes using synthetic human c-Ha-ras fragments containing 8-OH-G in various positions in hypermutagenic codon 12, including a tandem arrangement of two lesions (Jaoszyski et al. 2003). We demonstrated that in this experimental system Pol was much more efficient than DNA polymerases and ß, and even two adjacent 8-OH-G residues were efficiently bypassed by the enzyme. Moreover, Pol incorporated dAMP opposite 8-OH-G, showed a moderate ‘action-at-a-distance’ mutagenic effect, being able to misread guanine 3'-flanked by 8-OH-G, and was found to be strongly mutagenic in the tandem 8-OH-G system, where all four dNTPs were incorporated; dCMP and dAMP with very high, dGMP with moderate and dTMP with low efficiency (Jaoszyski et al. 2003). Polymerase (Pol), in turn, is known to bypass 1,N⁶-ethenoadenine in a relatively inefficient way, generating replication errors (Levine et al. 2001). This enzyme also catalyzes error-prone replication past acetylaminofluorene-derived lesions (Suzuki et al. 2001), abasic sites (Ohashi et al. 2000; Zhang et al. 2000b) and 8-OH-G (Zhang et al. 2000b). Pol turned out to be unable to bypass thymine-thymine dimers (both cis-syn cyclobutane dimers and 6-4 photoproducts) (Ohashi et al. 2000; Zhang et al. 2000b), and cisplatin DNA adducts (Ohashi et al. 2000; Gerlach et al. 2001). A highly interesting finding was the arylhydrocarbon receptor-dependent transcription of Pol (Ogi et al. 2001). This observation was subsequently followed by the discovery that Pol was able preferentially to incorporate the correct base opposite dG-N2-benzo[a]pyrene diolepoxide (Suzuki et al. 2001).

A large body of evidence supports the hypothesis that reactive oxygen species take part in activation of Ha-ras genes by inducing single substitutions in particular codons. High frequencies of GT transversion were observed in hypermutagenic codons 12 and 61 of the c-Ha-ras gene in some types of cancer, e.g. in squamous cell and basal cell carcinomas (White & Balmain 1988; van der Schoef et al. 1990; Pierceall et al. 1991). This particular substitution is the most frequent mutation caused by 8-OH-G (Shibutani et al. 1991; Cheng et al. 1992), a marker of DNA oxidation (Kamiya 2003). Indeed, a ras gene containing 8-OH-G in codon 12 was found to have transforming activity in NIH3T3 cells (Kamiya et al. 1992a). Therefore, systems containing 8-OH-G residues in mutation hotspots of ras genes have been established and widely used to study the bases of mutagenesis and proto-oncogene activating events (Kamiya et al. 1992b, 1995a,b; Le Page et al. 1995; Tan et al. 1999; Jaoszyski et al. 2003). In the previous studies (Jaoszyski et al. 2003), we applied an experimental system containing a single moiety of 8-OH-G in various positions of codon 12 of the human c-Ha-ras gene, or two lesions in a tandem arrangement, to characterize in detail the mutagenicity of Pol. Comparison with other DNA polymerases suggested the possible role of Pol in error-prone translesion DNA synthesis past 8-OH-G when two 8-OH-Gs were present in codon 12. This enzyme was not blocked or even inhibited by such a double lesion, in contrast to polymerases and ß (Jaoszyski et al. 2003). The present study explores in detail kinetics of translesion DNA synthesis past 8-OH-G by DNA polymerase , so far not investigated in a quantitative manner. We used the ras-gene experimental system for systematic study of the effects of 8-OH-G on DNA synthesis catalyzed by Pol. As mentioned above, this interesting enzyme was found to generate replication errors when its substrate contains a single 8-OH-G (Zhang et al. 2000b). However, the kinetics of this reaction, especially with DNA sequences corresponding to proto-oncogenes, remained unstudied. To estimate sequence-context effects, we compared the mutagenic potentials of two sequences: the previously used human c-Ha-ras and newly synthesized mouse Ha-ras1 fragments, differing in the sequence of the critical codon 12, but both being sensitive to oxidation of guanine. We analyzed four experimental systems: (i) direct mutagenesis system, the first replicated nucleotide was 8-OH-G 3'-flanked by G; (ii) ‘action-at-a-distance’ mutagenesis system, the first replicated nucleotide was G 3'-flanked by 8-OH-G; (iii) tandem lesion system, the first replicated nucleotide was 8-OH-G 3'-flanked by 8-OH-G; and (iv) control system with non-modified templates. We demonstrated that Pol was capable of an ‘action-at-a-distance’ mutagenesis, and this enzyme appeared to be the most mutagenic among Y-polymerases.

	Results

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Mouse Ha-ras1 sequence

To compare reaction rates between human and mouse ras sequences, we used the same experimental conditions (i.e., enzyme activity, substrate concentrations, reaction time and temperature) for both c-Ha-Ras and Ha-ras1 fragments. The sequences used and typical insertion reaction results are shown, respectively, in Figs 1 and 2.

View larger version (37K): [in this window] [in a new window]   Figure 1  Oligonucleotides corresponding to the region of the human c-Ha-ras (H) and mouse Ha-ras1 (M) genes used in the study. (A) Templates and primers, the position of codon 12 is indicated. (B) Experimental systems used in the insertion assay; only codon 12 of the templates is shown.

View larger version (43K): [in this window] [in a new window]   Figure 2  Representative example of the insertion assay. Reaction mixtures containing the mouse Ha-ras1 gene fragment annealed to the primer MP and increasing concentrations of one of four dNTPs were incubated with Pol. Products were separated on polyacrylamide gel, as described in Experimental procedures. Codon 12 is underlined. (A) Replication of unmodified control template M0. (B) replication of template M1 containing 8-OH-G in the first position of codon 12. (C) Replication of template M2 containing G in the first position of codon 12, and 8-OH-G in the second position. (D) Replication of template M3 containing two adjacent 8-OH-G in codon 12.

View this table: [in this window] [in a new window]   Table 1 Kinetic parameters of insertion reactions catalyzed by DNA polymerase

The insertion of dCMP catalyzed by Pol on the control template H0 (Fig. 3E, Table 1) was not accompanied by any misincorporation, but the efficiency of this reaction turned out to be over 31 times lower than the corresponding value obtained in the mouse system. Qualitatively, the effects observed in the case of sequences containing 8-OH-G were similar to those observed in the mouse fragments, but the reaction efficiencies were much lower. Gradually increasing inhibition of incorporation of dCMP was noticed, from k_cat/K_m = 5.32 in the case of the control template H0, through 1.31 and 1.15, respectively, in the direct mutagenesis system (template H1; Fig. 3F, Table 1) and ‘action-at-a-distance’ system (template H2; Fig. 3G, Table 1), up to 0.60 for two 8-OH-G residues in a tandem arrangement (template H3; Fig. 3H, Table 1). Direct mutagenesis (Fig. 3F), appearing as exclusive misincorporation of dAMP, was nearly 2.8 times more efficient than the correct insertion of dCMP. This is comparable to the three-fold difference obtained in the mouse system. Induction of mutagenesis by 8-OH-G 3'-flanking replicated, unmodified guanine (Fig. 3G) was barely within detection limits; misincorporation of dAMP was slightly over 10 times less efficient than that of dCMP. The misincorporation rate slightly increased when the first replicated nucleotide was 8-OH-G and the lesion was also present in the 3'-flanking position (Fig. 3H).

View larger version (30K): [in this window] [in a new window]   Figure 3  Single nucleotide insertion curves. Individual reaction mixtures containing template:primer substrate and increasing concentrations of one dNTP were incubated with Pol. Reaction products were separated on polyacrylamide gel, and the percent of extended primer was calculated from the band intensity. A non-linear fit was applied to obtain rectangular hyperbolic insertion curves, as described in Experimental procedures. Mean values from two to five independent experiments are shown. Bars represent standard deviations. The position of codon 12 is underlined. Replication of unmodified control templates: (A) mouse Ha-ras1 M0 and (E) human c-Ha-ras H0; replication in the direct mutagenesis system with 8-OH-G in the first position of codon 12: (B) mouse Ha-ras1 M1 and (F) human c-Ha-ras H1; ‘action-at-a-distance’ mutagenesis system with G in the first position of codon 12 and 8-OH-G in the second position: (C) mouse Ha-ras1 M2 and (G) human c-Ha-ras H2. Replication in the tandem system with two adjacent 8-OH-G in codon 12: (D) mouse Ha-ras1 M3 and (H) human c-Ha-ras H3.

	Discussion

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Jaoszyski et al. 2003

Jaoszyski et al. 2003

Jaoszyski et al. 2003

We observed a remarkable difference in the reaction efficiencies with the mouse and human sequences. Pol catalyzed incorporation much more efficiently on mouse templates, whereas human DNA fragments were distinctly worse substrates. There was only a four-base difference between the studied sequences. However, the mouse Ha-ras1 as compared to human c-Ha-ras has A instead of C in the third position of the codon 12, and this critical codon is 5'-flanked by T, instead of C. This remarkably increases the content of pyrimidines in the closest proximity of the first replicated nucleotide, and breaks the long sequence of purines present in the human fragment. Overall increase in the insertion efficiency observed in the mouse sequence may be due to the pyrimidine-rich neighborhood of the reaction site. However, the actual reason of such a difference in the insertion efficiency remains unknown. Regardless the differences, in the direct mutagenesis systems, the preference for dAMP over dCMP was nearly the same both on the mouse and human template, i.e., 3-fold and 2.8-fold, respectively. 8-OH-G in this system seems to be mutagenic to a similar degree, regardless of the overall efficiency of incorporation.

Interestingly, in direct mutagenesis experiments, Pol was found to be more mutagenic than Pol analyzed previously. The latter enzyme in the human c-Ha-Ras system inserted dCMP more efficiently than dAMP (Jaoszyski et al. 2003). In the present study, Pol incorporated dAMP with much higher efficiency than the correct dCMP. Moreover, Pol caused indirect mispairing (often referred to as ‘action-at-a-distance’ mutagenesis (Efrati et al. 1999)). This effect was barely noticeable in the human system, but very distinct when templates were mouse Ha-ras1 gene fragments. This difference is likely caused by the template-primer slipped misalignment. The 5'-templating base in the human system is C, whereas in the mouse sequence it is T. Thus, more efficient misincorporation of dAMP on M2 template is likely induced on the way of misalignment, and incorporation of dAMP opposite the next T. Since the base 5'-flanking the first replicated nucleotide on the template H2 is C, dAMP misinsertion is not favored. The ability of Pol to template-primer misalignment was shown by Wolfle et al. (2003). ‘Action-at-a-distance’ mutagenesis was reported for the first time by Kuchino et al. (1987) and concerned the Klenow fragment of bacterial polymerase I. Later, the eukaryotic repair DNA polymerase ß was also found to show a similar effect (Efrati et al. 1999), and recently we showed that 8-OH-G stimulated mispairing at a neighboring template site when replicated by Pol (Jaoszyski et al. 2003). Pol is the second polymerase belonging to the Y-family to show the same effect. It remains to be studied whether other enzymes of this group behave in a similar way.

Two adjacent 8-OH-G residues in codon 12, though inhibiting Pol, also induced misincorporation. However, this effect was not comparable to the strong relaxation of enzyme specificity observed previously in the case of Pol (Jaoszyski et al. 2003). This fact represents the most important qualitative difference in the actions of these two, otherwise similar, DNA polymerases catalyzing DNA synthesis past 8-OH-G. The mechanisms of replication across such a double lesion may be significantly different for various enzymes belonging to the Y-family. The presence of two guanines sensitive to oxidation in codon 12 of the c-Ha-Ras gene was postulated as an important fact supporting the role of reactive oxygen species in activation of the gene. Taking into account the data presented herein, Pol may rather play a role in direct mutagenic events, when a single 8-OH-G moiety is replicated and dAMP is misincorporated with high efficiency. Pol remains the main suspect when a tandem lesion is present and other DNA polymerases may be unable to pass such a distortion in the DNA chain.

	Experimental procedures

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Oligonucleotides corresponding to the codon 12 region of the human c-Ha-ras and mouse Ha-ras1 genes were synthesized by the Hokkaido System Science, Japan. The sequences of templates and primers are shown in Fig. 1. Human Pol protein was purified in the form of PolC (560 amino acids), which lacks motifs VIIa and VIIb that denote zinc clusters from the intact protein (870 amino acids), as previously described (Ohashi et al. 2000). Kinetic parameters were estimated as previously described (Jaoszyski et al. 2003). Briefly, 1 nM of Pol was incubated with ³²P-labeled primer:template substrate (20 nM) and increasing concentrations (0–500 µM) of a single deoxynucleotide (dCTP, dGTP, dATP or dTTP) in 40 mM Tris-HCl buffer (pH 8.0) containing 5 mM MgCl₂, 10 mM dithiothreitol, 60 nM KCl, 250 µg/mL BSA and 2.5% glycerol. Reaction mixtures were incubated at 30 °C for 1 min in a total volume of 5 µL, heated to 90 °C to inactivate the enzyme and then subjected to 20% polyacrylamide gel electrophoresis. Bands in gels were visualized and their intensities were measured using a BAS 2000 scanner (Fuji, Japan). The percentage of primer extended was plotted as a function of the dNTP concentration, and the kinetic parameters were calculated from the non-linear regression fit to a rectangular hyperbola (v = (V_max x [dNTP]/[K_m + [dNTP]), as described (Creighton et al. 1995). Average values obtained from two to five independent experiments are presented.

	Acknowledgements

 
This research was partially supported by a Grant-in-aid (No-13214049 to H. O.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Footnotes

 
Communicated by: Fumio Hanaoka

^aPresent address: Institute of Human Genetics, Polish Academy of Sciences, Strzeszyñska 32, 60-479 Pozna, Poland

^* Correspondence: E-mail: japawel{at}man.poznan.pl

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Received: 29 December 2004
Accepted: 2 March 2005

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