Synthesis of Thymine Glycol Containing Oligonucleotides from a Building Block with the Oxidized Base

Iwai, Shigenori

doi:10.1002/1521-3773(20001103)39:21<3874::aid-anie3874>3.0.co;2-o

Cited by 39 publications

(40 citation statements)

References 36 publications

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“…DNA oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis (DPAGE). Five pmol of each primer was 5Ј-end-labeled with T4 polynucleotide kinase (Invitrogen) in the presence of [␥- 32 P]ATP, and the unincorporated radiolabel was removed using a Sephadex G-25 spin column equilibrated with STE (100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM Na 2 EDTA). Primers were annealed to template strands in a stoichiometric ratio of 1:1.5 (primer-template) by heating (90°C, 5 min, 1ϫ STE buffer) and cooling on the bench top (15 min).…”

Section: Methodsmentioning

confidence: 99%

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Human DNA Polymerase κ Bypasses and Extends beyond Thymine Glycols during Translesion Synthesis in Vitro, Preferentially Incorporating Correct Nucleotides

Fischhaber

Gerlach

Feaver

et al. 2002

Journal of Biological Chemistry

104

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Human polymerase (pol), the product of the human POLK (DINB1) gene, is a member of the Y superfamily of DNA polymerases that support replicative bypass of chemically modified DNA bases (Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo, T., Walker, G. C., Wang, Z., and Woodgate, R. (2001) Mol. Cell 8, 7-8; Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V., and Friedberg, E. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11922-11927). Pol is shown here to bypass 5,6-dihydro-5,6-dihydroxythymine (thymine glycol) generated in two different DNA substrate preparations. Pol inserts the correct base adenine opposite thymine glycol in preference to the other three bases. Additionally, the enzyme correctly extends beyond the site of the thymine glycol lesion when presented with adenine opposite thymine glycol at the primer terminus. However, steady state kinetic analysis of nucleotides incorporated opposite thymine glycol demonstrates different misincorporation rates for guanine with each of the two DNA substrates. The two substrates differ only in the relative proportions of thymine glycol stereoisomers, suggesting that pol distinguishes among stereoisomers and exhibits reduced discrimination between purines when incorporating a base opposite a 5R thymine glycol stereoisomer. When extending beyond the site of the lesion, the misincorporation rate of pol for each of the three incorrect nucleotides (adenine, guanine, and thymine) is dramatically increased. Our findings suggest a role for pol in both nonmutagenic and mutagenic bypass of oxidative damage.Many types of base damage in DNA cause structural modifications that can result in the stalling or complete arrest of high fidelity DNA synthesis during DNA replication (3, 4). However, the potential for cell death attendant on arrested DNA replication can be mitigated by a mechanism called translesion DNA synthesis (TLS) (5-7). This process effects the replicative bypass of sites of base damage, allowing high fidelity semiconservative DNA synthesis to continue. Important new insights into the biochemical mechanism of TLS have recently been gained by the discovery of a number of new DNA polymerases, all of which share the properties of limited fidelity and processivity when copying undamaged DNA, as well as a lack of 3Ј 3 5Ј proofreading exonuclease activity (1, 5-9). Multiple DNA polymerases of this class have been shown to support TLS of one or more types of base damage in vitro. In some instances, this role is supported by genetic or other biological evidence. Hence, a general theme is beginning to emerge that the redundancy for error-prone DNA polymerases in prokaryotic and especially in eukaryotic cells reflects a requirement for the bypass of multiple types of base damage that can arrest normal DNA replication (5). Recent structural studies on a number of these polymerases suggest that translesion synthesis is effected by a less ...

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Section: Methodsmentioning

confidence: 99%

“…Both procedures result in a mixture of the four possible stereoisomers of Tg in different relative proportions (Fig. 1B) (32,33). We show by both qualitative and quantitative steady-state kinetic analysis that pol supports TLS across both of these substrates.…”

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confidence: 99%

Human DNA Polymerase κ Bypasses and Extends beyond Thymine Glycols during Translesion Synthesis in Vitro, Preferentially Incorporating Correct Nucleotides

Fischhaber

Gerlach

Feaver

et al. 2002

Journal of Biological Chemistry

104

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“…11-mers with the same sequence, were used for two reasons. One reason was that a tetramer could not be the substrate for uracil DNA glycosylase in the preparation of the AP site-containing oligonucleotide, and the other reason was that relatively long sequences were favorable at the deprotection step in our synthesis of the thymine glycol-containing oligomers (26). A dinucleoside monophosphate and a 30-mer containing the (6-4) photoproduct were used for the NMR experiments and the biochemical assays, respectively, as described in the following sections.…”

Section: Design and Preparation Of Oligonucleotides-mentioning

confidence: 99%

Identification and Characterization of an Intermediate in the Alkali Degradation of (6-4) Photoproduct-containing DNA

Higurashi

Ohtsuki

Inase

et al. 2003

Journal of Biological Chemistry

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The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4)TC), was prepared, and its degradation in 50 mM KOH at 60°C was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was UV light in solar radiation is absorbed by nucleobases in DNA and induces photochemical reactions. The major forms of DNA damage by UV radiation are the cis-syn cyclobutane pyrimidine dimer (CPD) 1 and the pyrimidine(6-4)pyrimidone photoproduct ((6-4) photoproduct) formed between adjacent pyrimidine bases (1). The formation of these lesions not only induces mutations of genetic information (2) but also alters the chemical stability of DNA. In the case of the CPD formed at the sequences containing cytosine, the hydrolysis rate of the amino function is much higher than that of the normal cytosine base (3), which results in the C3 T transition (4 -6). The (6-4) photoproduct was originally identified as a UV-induced alkalisensitive lesion (7,8). Strand breaks occur at the sites of this photoproduct when UV-irradiated DNA is treated with hot alkali, and this procedure has been used to map the (6-4) photoproduct in defined DNA sequences at nucleotide resolution (9 -12). This alkali lability has also been applied for footprinting experiments to analyze protein-DNA interactions in vivo (13,14). While the reactions of other alkali-labile DNA damages such as the apurinic/apyrimidinic (AP) site (15) and the bleomycin-induced lesion (16) have been analyzed in detail, the mechanism of the strand break caused at the (6-4) photoproduct by the alkali treatment has not been elucidated so far. Franklin et al. (8) suggested the hydrolysis of the glycosyl bond of the 3Ј component of this photoproduct, but there have been no subsequent reports.The (6-4) photoproduct is reportedly more mutagenic than the CPD (17-19) and blocks replication without being bypassed by error-free translesion synthesis, whereas DNA polymerase correctly incorporates dATP opposite the CPD formed at the TT sequence (20, 21). To avoid mutagenesis and carcinogenesis by the (6-4) photoproduct, this damage must be removed by the nucleotide excision repair (NER) pathway in cells (22)(23)(24). No base excision repair (BER) enzyme is known for the (6-4) photoproduct, but if the photoproduct can be degraded artificially, this DNA may be repaired by the BER pathway, even in xeroderma pigmentosum patients lacking NER function. For such an application, it is important to characterize the chemical properties of the (6-4) photoproduct.In this paper we describe the analysis of the alkali degradation of DNA containing the (6-4) photoproduct using chemically synthesized oligonucleotides. A relati...

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“…In this regard, Iwai (33,34) described the synthesis of a building block of thymidine glycol where the nucleobase hydroxyl group(s) were protected with tert-butyldimethylsilyl (TBDMS) group. Very recently, Cadet et al (35) reported the synthesis of a Tg building block where the nucleobase hydroxyl group(s) was protected with a base-labile levulinyl functionality.…”

Section: Introductionmentioning

confidence: 99%

“…Very recently, Cadet et al (35) reported the synthesis of a Tg building block where the nucleobase hydroxyl group(s) was protected with a base-labile levulinyl functionality. On the other hand, the deprotection of ODNs synthesized from the commercially available building block of 8-oxodG requires prolonged treatment with concentrated ammonium hydroxide; during such treatment, thymidine glycol, however, was shown to be significantly degraded (33). Therefore, dual insertion of 8-oxodG and thymidine glycol necessitates alternative synthetic strategies.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Thermodynamic Studies of Oligodeoxyribonucleotides Containing Tandem Lesions of Thymidine Glycol and 8-Oxo-2‘-deoxyguanosine

Wang

2006

Chem. Res. Toxicol.

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Thymidine glycol (Tg), which is also known as 5,6-dihydroxy-5,6-dihydrothymidine, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) are two major types of DNA damage products induced by reactive oxygen species (ROS). Here we report the synthesis of oligodeoxyribonucleotides (ODNs) containing both Tg and 8-oxodG. The dual incorporation of the two single-base lesions was achieved by using a phosphoramidite building block of 8-oxodG with ultramild base protecting group and a building block of Tg whose nucleobase hydroxyl groups were protected with acetyl functionality. The availability of ODNs carrying neighboring 8-oxodG and Tg provided authentic substrates for assessing the formation and examining the replication and repair of this kind of tandem lesions. In addition, thermodynamic parameters derived from melting temperature data revealed that tandem lesions destabilized the double helix to a greater extent than either of the two single-base lesions alone. The thermodynamic results could offer a basis for understanding the repair of the tandem base lesions.

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Synthesis of Thymine Glycol Containing Oligonucleotides from a Building Block with the Oxidized Base

Cited by 39 publications

References 36 publications

Human DNA Polymerase κ Bypasses and Extends beyond Thymine Glycols during Translesion Synthesis in Vitro, Preferentially Incorporating Correct Nucleotides

Human DNA Polymerase κ Bypasses and Extends beyond Thymine Glycols during Translesion Synthesis in Vitro, Preferentially Incorporating Correct Nucleotides

Identification and Characterization of an Intermediate in the Alkali Degradation of (6-4) Photoproduct-containing DNA

Synthesis and Thermodynamic Studies of Oligodeoxyribonucleotides Containing Tandem Lesions of Thymidine Glycol and 8-Oxo-2‘-deoxyguanosine

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