Articles pubs.acs.org/acschemicalbiology
O6‑Alkylguanine Postlesion DNA Synthesis Is Correct with the Right Complement of Hydrogen Bonding Hailey L. Gahlon, Melissa L. Boby, and Shana J. Sturla* Department of Health Sciences and Technology, Institute of Food Nutrition and Health, ETH Zürich, Schmelzbergstrasse 9, 8092 Zürich, Switzerland S Supporting Information *
ABSTRACT: The ability of a DNA polymerase to replicate DNA beyond a mismatch containing a DNA lesion during postlesion DNA synthesis (PLS) can be a contributing factor to mutagenesis. In this study, we investigate the ability of Dpo4, a Y-family DNA polymerase from Sulfolobus solfataricus, to perform PLS beyond the pro-mutagenic DNA adducts O6benzylguanine (O6-BnG) and O6-methylguanine (O6-MeG). Here, O6-BnG and O6-MeG were paired opposite artificial nucleosides that were structurally altered to systematically test the influence of hydrogen bonding and base pair size and shape on O6-alkylguanine PLS. Dpo4-mediated PLS was more efficient past pairs containing Benzi than pairs containing the other artificial nucleoside probes. Based on steady-state kinetic analysis, frequencies of mismatch extension were 7.4 × 10−3 and 1.5 × 10−3 for Benzi:O6-MeG and Benzi:O6-BnG pairs, respectively. Correct extension was observed when O6-BnG and O6-MeG were paired opposite the smaller nucleoside probes Benzi and BIM; conversely, Dpo4 did not extend past the larger nucleoside probes, Peri and Per, placed opposite O6-BnG and O6-MeG. Interestingly, Benzi was extended with high fidelity by Dpo4 when it was paired opposite O6-BnG and O6-MeG but not opposite G. These results indicate that hydrogen bonding is an important noncovalent interaction that influences the fidelity and efficiency of Dpo4 to perform high-fidelity O6-alkylguanine PLS. lthough high fidelity DNA polymerases contain stringent active sites and inefficiently replicate past structurally perturbed base pairs,1 error-prone DNA polymerases (Yfamily) contain spacious active sites that can accommodate DNA adducts, enabling them to perform translesion DNA synthesis (TLS).2−4 This lesion bypass is important to understand in the context of how mutations arise at sites containing chemically modified DNA. As compared to TLS, error-prone DNA polymerases can be less effective at mismatch extension or postlesion DNA synthesis (PLS). Exceptions include human Pol κ and human Pol ι, which efficiently catalyze extension from most canonical DNA terminal mismatches.5,6 Also, in studies involving Arabidopsis Pol κ that contained a truncated C-terminal domain, an enhanced ability to extend past 8-oxoguanine mismatches was observed.7 Mismatches can destabilize base stacking interactions in DNA and RNA, and the greatest distortions occur with purine:purine or pyrimidine:pyrimidine mismatches.8,9 Usually, replicative DNA polymerases are inefficient at mismatch extension due to their tight active sites that can sense geometrical base pairing perturbations.10 In contrast, Y-family DNA polymerases operate more efficiently for extension beyond mismatches due to more relaxed active sites. The archeal Y-family DNA polymerase Dpo4 extends from all canonical DNA mismatches; however, extension from a G:T mismatch is inefficient, and based on X-ray structural analysis, a reverse wobble pair between G:T misorients the 3′-OH group
A
© 2014 American Chemical Society
on the primer strand, indicating that less efficient catalysis could occur.11 DNA polymerase-mediated extension beyond canonical nucleotide mismatches has been explored to a larger extent than for terminal mismatches containing a DNA lesion, i.e., PLS. It is suggested that a two polymerase mechanism can be evoked during synthesis past damaged DNA substrates, i.e. one polymerase for TLS and another polymerase for PLS. For PLS, the mammalian DNA polymerase zeta appears to play a role in extension beyond DNA lesions. Studies that interrogate mechanisms of TLS and PLS are needed to understand their role in mutagenesis and carcinogenesis. Nucleoside analogs have been used to interrogate TLS and elucidate chemical interactions that impact DNA replication on damaged DNA substrates.12−16 For O6-alkylguanine adducts, the use of synthetic nucleoside triphosphates in a TLS study showed how hydrogen bonding is important for the fidelity observed by Dpo4 to bypass O6-MeG.17 Further, nucleoside analogs with aromatic hydrocarbon functionality can stabilize duplexes containing O6-alkylguanine DNA adducts through base stacking interactions.18,19 Recently, we communicated that the rates and fidelities of Dpo4-mediated synthesis past terminal base pairs containing O6-BnG placed opposite of Received: May 27, 2014 Accepted: September 26, 2014 Published: September 26, 2014 2807
dx.doi.org/10.1021/cb500415q | ACS Chem. Biol. 2014, 9, 2807−2814
ACS Chemical Biology
Articles
alkylguanine adducts with synthetic nucleoside probes of varied hydrogen bonding capacities. In this study, we investigated Dpo4-mediated O6-alkylguanine PLS with a series of artificial nucleosides (Figure 1B) that vary in nucleobase size and the capacity to hydrogen bond with O6-alkylguanine. The structural features of these artificial nucleosides allowed for testing (1) the steric contraints within the active site of Dpo4 during PLS and (2) the influence of hydrogen bonding groups on the fidelity and efficiency of Dpo4 to extend past O6-BnG and O6-MeG. Primer extension studies were performed to investigate the ability of Dpo4 to synthesize past mismatches containing these artificial nucleosides as well as canonical nucleosides (A, T, C, and G) placed on the 3′-end of the primer and paired opposite G, O6-BnG, or O6-MeG (Figure 1A). Single nucleotide insertion studies were performed to determine Dpo4’s fidelity for O6-alkylguanine PLS, and steady-state kinetic experiments were performed to determine the catalytic efficiency of Dpo4 on G-, O6-BnG-, and O6-MeG-containing templates. Finally, computational modeling was done to investigate the potential base pairing interactions between O6-BnG and the artificial nucleosides within the active site of Dpo4.
nucleoside analogs differ depending on nucleobase size and shape for the nucleobase-surrogates BIM and Peri (Figure 1B)20. In this manner, we determined nucleobase size contstraints that promote correct Dpo4-mediated O6-BnG PLS.
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Figure 1. (A) Schematic of Dpo4-mediated O6-alkylguanine PLS. (B) Artificial nucleoside probes BIM, Benzi, Peri and Per used in this study. (C) DNA sequences of template and primer oligonucleotides (X = G, O6-BnG, or O6-MeG and N = BIM, Benzi, Peri, Per, or canonical bases A, T, C or G).
RESULTS AND DISCUSSION Pro-mutagenic O6-alkylguanine DNA adducts can be generated from the bioactivation of nitrosamines, e.g., N-methyl-Nbenzylnitrosamine, and can induce G to A transition mutations due to the high misincorporation of T during DNA polymerase-mediated TLS.26,27 While contributions of mutagenesis for TLS are well described, information is lacking regarding a molecular level understanding of mismatch extension by DNA polymerases, especially in the context of PLS. For this study, nucleoside analogs were used to define the manner in which the size and shape of the base pairs at the terminal position of DNA influence the ability of Dpo4 to replicate beyond O6-alkylguanine mismatches. The nucleoside probes were structurally varied in order to test systematically the impact of size, shape, and hydrogen bonding capacity on
Data regarding nucleotide insertion and extension by Dpo4 have increased our understanding regarding mechanisms of TLS and its contribution in mutagenesis.20−23 Specifically in the case of O6-alkylguanine, it has been shown that Dpo4 bypasses O6-MeG more efficiently than O6-BnG.24,25 While the fidelity and efficiency of Dpo4-mediated TLS on O6-alkylguaine DNA adducts is well described, information is lacking that addresses the impact of alkylation size, e.g., methyl vs benzyl, on O6-alkylguanine PLS, as well as the interaction of O6-
Figure 2. (A) DNA sequence used for Dpo4-mediated primer extension analysis (X = G, O6-MeG, or O6-BnG and N = Peri, Per, BIM, or Benzi). The extension by Dpo4 past the BIM (B), Peri (C), Benzi (D), and Per (E) nucleoside analogs. Final dNTP concentrations were 100 μM. B, Blank; 4, all four dNTPs; G, dGTP; T, dTTP; C, dCTP. 2808
dx.doi.org/10.1021/cb500415q | ACS Chem. Biol. 2014, 9, 2807−2814
ACS Chemical Biology
Articles
Figure 3. (A) DNA sequence used for Dpo4-mediated primer extension analysis (X = G, O6-MeG, or O6-BnG and N = C, A, T, or G). The extension by Dpo4 past canonical C (B), canonical G (C), canonical T (D), and canonical A (E). Final dNTP concentrations were 100 μM. B, Blank; 4, all four dNTPs; G, dGTP; T, dTTP; C, dCTP.
Dpo4-mediated O6-alkylguanine PLS. BIM and Benzi contain benzene-derived bicyclic ring systems, while Peri and Per contain larger naphthyl-derived tricyclic moieties (Figure 1B). Benzi and Per contain a carbonyl hydrogen bond acceptor and an amino N−H hydrogen bond donor. In contrast, BIM and Peri contain an imidazole nitrogen hydrogen bond acceptor. With the use of adduct-specific nucleoside probes, we were able to demonstrate that hydrogen bonding is an important noncovalent interaction that facilitates correct O6-alkylguanine PLS by Dpo4. Dpo4-Extension from Primer Template Termini with Artificial Primers. Dpo4-mediated extension of 24-mer primers containing BIM, Benzi, Peri, and Per (Figure 1C) placed opposite 28-mer templates containing G, O6-MeG, or O6-BnG (Figure 1C) was examined (Figure 2). Phosphoramidites of BIM, Benzi, Peri, and Per were prepared and incorporated into DNA via solid-phase synthesis as previously described. 20,28 Single nucleotide insertion studies (i.e., incubation with one dNTP) and full length extension analysis (i.e., incubation with all four canonical dNTPs) were performed. Throughout this text, mismatches are designated as N:X pairs with the first base (here N) deriving from the primer strand and the second (here X) from the template (Figure 1C). The probes Peri and Per placed opposite O6-BnG and O6MeG impeded Dpo4 from catalyzing appreciable extension (Figure 2C and E, respectively), except in the case of Peri:O6MeG, for which slight insertion of dGMP (
O6‑Alkylguanine Postlesion DNA Synthesis Is Correct with the Right Complement of Hydrogen Bonding Hailey L. Gahlon, Melissa L. Boby, and Shana J. Sturla* Department of Health Sciences and Technology, Institute of Food Nutrition and Health, ETH Zürich, Schmelzbergstrasse 9, 8092 Zürich, Switzerland S Supporting Information *
ABSTRACT: The ability of a DNA polymerase to replicate DNA beyond a mismatch containing a DNA lesion during postlesion DNA synthesis (PLS) can be a contributing factor to mutagenesis. In this study, we investigate the ability of Dpo4, a Y-family DNA polymerase from Sulfolobus solfataricus, to perform PLS beyond the pro-mutagenic DNA adducts O6benzylguanine (O6-BnG) and O6-methylguanine (O6-MeG). Here, O6-BnG and O6-MeG were paired opposite artificial nucleosides that were structurally altered to systematically test the influence of hydrogen bonding and base pair size and shape on O6-alkylguanine PLS. Dpo4-mediated PLS was more efficient past pairs containing Benzi than pairs containing the other artificial nucleoside probes. Based on steady-state kinetic analysis, frequencies of mismatch extension were 7.4 × 10−3 and 1.5 × 10−3 for Benzi:O6-MeG and Benzi:O6-BnG pairs, respectively. Correct extension was observed when O6-BnG and O6-MeG were paired opposite the smaller nucleoside probes Benzi and BIM; conversely, Dpo4 did not extend past the larger nucleoside probes, Peri and Per, placed opposite O6-BnG and O6-MeG. Interestingly, Benzi was extended with high fidelity by Dpo4 when it was paired opposite O6-BnG and O6-MeG but not opposite G. These results indicate that hydrogen bonding is an important noncovalent interaction that influences the fidelity and efficiency of Dpo4 to perform high-fidelity O6-alkylguanine PLS. lthough high fidelity DNA polymerases contain stringent active sites and inefficiently replicate past structurally perturbed base pairs,1 error-prone DNA polymerases (Yfamily) contain spacious active sites that can accommodate DNA adducts, enabling them to perform translesion DNA synthesis (TLS).2−4 This lesion bypass is important to understand in the context of how mutations arise at sites containing chemically modified DNA. As compared to TLS, error-prone DNA polymerases can be less effective at mismatch extension or postlesion DNA synthesis (PLS). Exceptions include human Pol κ and human Pol ι, which efficiently catalyze extension from most canonical DNA terminal mismatches.5,6 Also, in studies involving Arabidopsis Pol κ that contained a truncated C-terminal domain, an enhanced ability to extend past 8-oxoguanine mismatches was observed.7 Mismatches can destabilize base stacking interactions in DNA and RNA, and the greatest distortions occur with purine:purine or pyrimidine:pyrimidine mismatches.8,9 Usually, replicative DNA polymerases are inefficient at mismatch extension due to their tight active sites that can sense geometrical base pairing perturbations.10 In contrast, Y-family DNA polymerases operate more efficiently for extension beyond mismatches due to more relaxed active sites. The archeal Y-family DNA polymerase Dpo4 extends from all canonical DNA mismatches; however, extension from a G:T mismatch is inefficient, and based on X-ray structural analysis, a reverse wobble pair between G:T misorients the 3′-OH group
A
© 2014 American Chemical Society
on the primer strand, indicating that less efficient catalysis could occur.11 DNA polymerase-mediated extension beyond canonical nucleotide mismatches has been explored to a larger extent than for terminal mismatches containing a DNA lesion, i.e., PLS. It is suggested that a two polymerase mechanism can be evoked during synthesis past damaged DNA substrates, i.e. one polymerase for TLS and another polymerase for PLS. For PLS, the mammalian DNA polymerase zeta appears to play a role in extension beyond DNA lesions. Studies that interrogate mechanisms of TLS and PLS are needed to understand their role in mutagenesis and carcinogenesis. Nucleoside analogs have been used to interrogate TLS and elucidate chemical interactions that impact DNA replication on damaged DNA substrates.12−16 For O6-alkylguanine adducts, the use of synthetic nucleoside triphosphates in a TLS study showed how hydrogen bonding is important for the fidelity observed by Dpo4 to bypass O6-MeG.17 Further, nucleoside analogs with aromatic hydrocarbon functionality can stabilize duplexes containing O6-alkylguanine DNA adducts through base stacking interactions.18,19 Recently, we communicated that the rates and fidelities of Dpo4-mediated synthesis past terminal base pairs containing O6-BnG placed opposite of Received: May 27, 2014 Accepted: September 26, 2014 Published: September 26, 2014 2807
dx.doi.org/10.1021/cb500415q | ACS Chem. Biol. 2014, 9, 2807−2814
ACS Chemical Biology
Articles
alkylguanine adducts with synthetic nucleoside probes of varied hydrogen bonding capacities. In this study, we investigated Dpo4-mediated O6-alkylguanine PLS with a series of artificial nucleosides (Figure 1B) that vary in nucleobase size and the capacity to hydrogen bond with O6-alkylguanine. The structural features of these artificial nucleosides allowed for testing (1) the steric contraints within the active site of Dpo4 during PLS and (2) the influence of hydrogen bonding groups on the fidelity and efficiency of Dpo4 to extend past O6-BnG and O6-MeG. Primer extension studies were performed to investigate the ability of Dpo4 to synthesize past mismatches containing these artificial nucleosides as well as canonical nucleosides (A, T, C, and G) placed on the 3′-end of the primer and paired opposite G, O6-BnG, or O6-MeG (Figure 1A). Single nucleotide insertion studies were performed to determine Dpo4’s fidelity for O6-alkylguanine PLS, and steady-state kinetic experiments were performed to determine the catalytic efficiency of Dpo4 on G-, O6-BnG-, and O6-MeG-containing templates. Finally, computational modeling was done to investigate the potential base pairing interactions between O6-BnG and the artificial nucleosides within the active site of Dpo4.
nucleoside analogs differ depending on nucleobase size and shape for the nucleobase-surrogates BIM and Peri (Figure 1B)20. In this manner, we determined nucleobase size contstraints that promote correct Dpo4-mediated O6-BnG PLS.
■
Figure 1. (A) Schematic of Dpo4-mediated O6-alkylguanine PLS. (B) Artificial nucleoside probes BIM, Benzi, Peri and Per used in this study. (C) DNA sequences of template and primer oligonucleotides (X = G, O6-BnG, or O6-MeG and N = BIM, Benzi, Peri, Per, or canonical bases A, T, C or G).
RESULTS AND DISCUSSION Pro-mutagenic O6-alkylguanine DNA adducts can be generated from the bioactivation of nitrosamines, e.g., N-methyl-Nbenzylnitrosamine, and can induce G to A transition mutations due to the high misincorporation of T during DNA polymerase-mediated TLS.26,27 While contributions of mutagenesis for TLS are well described, information is lacking regarding a molecular level understanding of mismatch extension by DNA polymerases, especially in the context of PLS. For this study, nucleoside analogs were used to define the manner in which the size and shape of the base pairs at the terminal position of DNA influence the ability of Dpo4 to replicate beyond O6-alkylguanine mismatches. The nucleoside probes were structurally varied in order to test systematically the impact of size, shape, and hydrogen bonding capacity on
Data regarding nucleotide insertion and extension by Dpo4 have increased our understanding regarding mechanisms of TLS and its contribution in mutagenesis.20−23 Specifically in the case of O6-alkylguanine, it has been shown that Dpo4 bypasses O6-MeG more efficiently than O6-BnG.24,25 While the fidelity and efficiency of Dpo4-mediated TLS on O6-alkylguaine DNA adducts is well described, information is lacking that addresses the impact of alkylation size, e.g., methyl vs benzyl, on O6-alkylguanine PLS, as well as the interaction of O6-
Figure 2. (A) DNA sequence used for Dpo4-mediated primer extension analysis (X = G, O6-MeG, or O6-BnG and N = Peri, Per, BIM, or Benzi). The extension by Dpo4 past the BIM (B), Peri (C), Benzi (D), and Per (E) nucleoside analogs. Final dNTP concentrations were 100 μM. B, Blank; 4, all four dNTPs; G, dGTP; T, dTTP; C, dCTP. 2808
dx.doi.org/10.1021/cb500415q | ACS Chem. Biol. 2014, 9, 2807−2814
ACS Chemical Biology
Articles
Figure 3. (A) DNA sequence used for Dpo4-mediated primer extension analysis (X = G, O6-MeG, or O6-BnG and N = C, A, T, or G). The extension by Dpo4 past canonical C (B), canonical G (C), canonical T (D), and canonical A (E). Final dNTP concentrations were 100 μM. B, Blank; 4, all four dNTPs; G, dGTP; T, dTTP; C, dCTP.
Dpo4-mediated O6-alkylguanine PLS. BIM and Benzi contain benzene-derived bicyclic ring systems, while Peri and Per contain larger naphthyl-derived tricyclic moieties (Figure 1B). Benzi and Per contain a carbonyl hydrogen bond acceptor and an amino N−H hydrogen bond donor. In contrast, BIM and Peri contain an imidazole nitrogen hydrogen bond acceptor. With the use of adduct-specific nucleoside probes, we were able to demonstrate that hydrogen bonding is an important noncovalent interaction that facilitates correct O6-alkylguanine PLS by Dpo4. Dpo4-Extension from Primer Template Termini with Artificial Primers. Dpo4-mediated extension of 24-mer primers containing BIM, Benzi, Peri, and Per (Figure 1C) placed opposite 28-mer templates containing G, O6-MeG, or O6-BnG (Figure 1C) was examined (Figure 2). Phosphoramidites of BIM, Benzi, Peri, and Per were prepared and incorporated into DNA via solid-phase synthesis as previously described. 20,28 Single nucleotide insertion studies (i.e., incubation with one dNTP) and full length extension analysis (i.e., incubation with all four canonical dNTPs) were performed. Throughout this text, mismatches are designated as N:X pairs with the first base (here N) deriving from the primer strand and the second (here X) from the template (Figure 1C). The probes Peri and Per placed opposite O6-BnG and O6MeG impeded Dpo4 from catalyzing appreciable extension (Figure 2C and E, respectively), except in the case of Peri:O6MeG, for which slight insertion of dGMP (
