Kinetic Analysis of the Coding Properties of O6-Methylguanine in DNA: The Crucial Role of the Conformation of the Phosphodiester Bond

Tan, Hwee-Boon; Swann, Peter F.; Chance, Edwin M.

doi:10.1021/bi00183a042

Cited by 53 publications

(51 citation statements)

References 48 publications

(72 reference statements)

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“…The highfidelity replicative DNA polymerases which depend strictly upon a proper W-C base pairing geometry synthesize DNA across the m6G and 8-oxoG lesions by incorporating a T or an A, respectively, and by extending therefrom. This is because an m6G · T mispair closely resembles a W-C base pair (8,18,23) and an 8-oxoG(syn) · A(anti) Hoogsteen base pair also has the geometric features that resemble a W-C base pair (1,7,12).…”

Section: Discussionmentioning

confidence: 99%

Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Johnson

Haracska

Prakash

et al. 2006

Molecular and Cellular Biology

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Human DNA polymerase (Pol ) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N 6 of an A or the O 6 of a G, here we provide evidence that whereas hydrogen bonding at N 6 is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O 6 is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O 6 and N7 hydrogen bonding to DNA synthesis by Pol , we have examined its proficiency for replicating through the 6 O-methyl guanine and 8-oxoguanine lesions, which affect the O 6 and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O 6 is an imperative. The dispensability of N 6 hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N 6 positions of templating A.Human DNA polymerase (Pol ), a member of the Y family of polymerases, differs from other DNA polymerases in that it incorporates nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines (4,6,19,21,26). Pol exhibits the highest efficiency and fidelity opposite template A, where it misincorporates nucleotides with frequencies of ϳ10 Ϫ4 to 10 Ϫ5 ; opposite template G, Pol incorporates a C with an efficiency that is 5-to 10-fold reduced compared to a T opposite template A, and a T is misincorporated opposite template G with an efficiency that is only a fewfold-lowered than that for a C. Pol 's efficiency and fidelity are much reduced opposite pyrimidine templates, particularly opposite template T (4, 6, 19, 21, 26).The ternary crystal structures of Pol with template A and incoming dTTP and with template G and incoming dCTP have shown that the purine template adopts a syn conformation in the Pol active site and forms a Hoogsteen base pair with the incoming nucleotide which remains in the anti conformation (14, 16). The binding of the pyrimidine nucleotide induces a conformational change in the template purine from anti to syn in the Pol active site, and that results because the active site can accommodate only the ϳ8.6-Å C 1 Ј-C 1 Ј distance of sugars in a Hoogsteen base pair but not the C 1 Ј-C 1 Ј distance of ϳ1...

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Section: Discussionmentioning

confidence: 99%

Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Johnson

Haracska

Prakash

et al. 2006

Molecular and Cellular Biology

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“…The NMR results and crystal structures demonstrate that the O 6 -methylG:T base pair forms a pseudo Watson-Crick pair, with a single hydrogen bond formed between the O2 atom of T and the N2 atom of O 6 -methylG (11,13,14,16). The shape of the Watson-Crick-like O 6 -methylG:T base pair fits in the active site of the DNA polymerase without distorting the DNA, thereby contributing to the incorporation of dTTP opposite the lesion (17). Also, the sequence context of O 6 -methylG has been shown to influence the extent of dTTP incorporation by DNA polymerases opposite the lesion (18).…”

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confidence: 97%

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

Pence

Choi²,

Egli

et al. 2010

Journal of Biological Chemistry

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O6 -Methylguanine (O 6 -methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase core enzyme was determined for nucleoside triphosphate incorporation opposite O 6 -methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol , which showed that dTTP incorporation occurs with high efficiency opposite O Alkylating agents damage DNA by reacting with the nitrogen and oxygen atoms in DNA bases. Human exposure to alkylating agents arises from endogenous (e.g. food-derived nitrosamines) and exogenous (e.g. tobacco-specific nitrosoamines and chemotherapeutic agents including temozolomide and streptozotocin) sources (1). The endogenously produced compound S-adenosylmethionine also methylates DNA (2). Many alkylating agents that react with DNA form the mutagenic and cytotoxic DNA lesion O 6 -alkylguanine (3), along with other methylated bases. The mutagenicity of O 6 -alkylated DNA is a factor in human diseases such as cancer, teratogenic defects, and premature aging (1). Work focused on the adduct O 6 -methylG 2 has shown that it causes G:C3 A:T transition mutations (4).The mutagenic potential of O -methylG:T base pair fits in the active site of the DNA polymerase without distorting the DNA, thereby contributing to the incorporation of dTTP opposite the lesion (17). Also, the sequence context of O 6 -methylG has been shown to influence the extent of dTTP incorporation by DNA polymerases opposite the lesion (18).Replicative DNA polymerases catalyze the misincorporation of dTTP opposite O 6 -alkylG with similar or even higher efficiency than incorporation of the correct dCTP (4,7,14,18). Similarly, the Y-family DNA polymerases , , and all show poor nucleotide discrimination when bypassing O 6 -alkylG (5, 8). Pols and have similar efficiencies for dTTP * This work was supported, in whole or in part, by National Institutes of Health Grants R01 ES010375 (to F. P. G.), P01 ES005355 (to M. E.), P30 ES000267 (to F. P. G. and M. E.), and T32 ES007028 (to F. P. G. and M. G. P.). Vanderbilt University is a member institution of LS-CAT at the Advanced Photon Source (APS), Argonne, Illinois. Use of the APS was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-06CH11357. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. The atomic coordinates and structure factors (codes 3NGD and 3OSN)

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“…Nevertheless, DNA polymerases incorporate T opposite m6G more often than C, because the m6G ⅐ T mispair retains the Watson-Crick geometry more closely than the m6G ⅐ C base pair. At neutral pH, C is inserted opposite m6G via a wobble configuration, but the phosphodiester links both 3Ј and 5Ј to the C are distorted in this base pair (18,19,24,40,41,46). m6G is a block to synthesis by prokaryotic and eukaryotic DNA polymerases.…”

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confidence: 99%

Replication past O⁶-Methylguanine by Yeast and Human DNA Polymerase η

Haracska

Prakash

2000

Molecular and Cellular Biology

133

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O6 -Methylguanine (m6G) is formed by the action of alkylating agents such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase (Pol) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in the rad30⌬ pol32⌬ double mutant of yeast, which lacks the RAD30 gene that encodes Pol and the Pol32 subunit of DNA polymerase ␦ (Pol␦). Although Pol␦ can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Pol is much more efficient in replicating through m6G than Pol␦. Both Pol and Pol␦ insert a C or a T residue opposite from m6G; Pol, however, is more accurate, as it inserts a C about twice as frequently as Pol␦. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Pol could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy. O6 -Methylguanine (m6G) is formed in DNA by treatment with alkylating agents such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG). m6G is highly mutagenic, and the mutagenic and carcinogenic potency of alkylating agents closely parallels their ability to form m6G in DNA (31). In yeast as well as higher eukaryotes, m6G specifically induces G ⅐ C to A ⅐ T transition mutations (30,35).Alkylation at the O 6 position of guanine has a profound effect on base pairing properties. Melting studies of DNA duplexes containing m6G have shown that the m6G ⅐ T base pair is energetically less stable than the m6G ⅐ C base pair (10), and nuclear magnetic resonance studies have indicated that the m6G ⅐ T base pair is less hydrogen bonded than the m6G ⅐ C base pair (33, 34). Nevertheless, DNA polymerases incorporate T opposite m6G more often than C, because the m6G ⅐ T mispair retains the Watson-Crick geometry more closely than the m6G ⅐ C base pair. At neutral pH, C is inserted opposite m6G via a wobble configuration, but the phosphodiester links both 3Ј and 5Ј to the C are distorted in this base pair (18,19,24,40,41,46). m6G is a block to synthesis by prokaryotic and eukaryotic DNA polymerases. Extensive steady-state kinetic analyses have indicated that the Escherichia coli Klenow fragment is inhibited by m6G both at the step of insertion of a nucleotide opposite the lesion and at the step of extension from the m6G ⅐ C or m6G ⅐ T base pair (7). Sequenase (T7 DNA polymerase) is partially inhibited by m6G at both these steps (42). Eukaryotic DNA polymerase ␣, required for lagging strand DNA synthesis, is strongly blocked one base before m6G, indicating an inhibition of nucleotide insertion opposite the lesion (42). m6G also blocks DNA polymerase ␤, which is involved i...

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Kinetic Analysis of the Coding Properties of O6-Methylguanine in DNA: The Crucial Role of the Conformation of the Phosphodiester Bond

Cited by 53 publications

References 48 publications

Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

Replication past O⁶-Methylguanine by Yeast and Human DNA Polymerase η

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Kinetic Analysis of the Coding Properties of O6-Methylguanine in DNA: The Crucial Role of the Conformation of the Phosphodiester Bond

Cited by 53 publications

References 48 publications

Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

Replication past O6-Methylguanine by Yeast and Human DNA Polymerase η

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Replication past O⁶-Methylguanine by Yeast and Human DNA Polymerase η