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. 2012 Mar;40(5):2357-67.
doi: 10.1093/nar/gkr980. Epub 2011 Nov 15.

Development of a 'clickable' non-natural nucleotide to visualize the replication of non-instructional DNA lesions

Edward A Motea  1 , Irene Lee, Anthony J Berdis
Affiliations

Development of a 'clickable' non-natural nucleotide to visualize the replication of non-instructional DNA lesions

Edward A Motea et al. Nucleic Acids Res. 2012 Mar.

Abstract

The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using 'click' chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.

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Figures

Figure 1.
Figure 1.
Non-natural nucleotides as probes for TLS. (A) Comparison of the structures for an abasic site with that for a tetrahydrofuran moiety, the stable and non-reactive mimetic for an abasic site. (B) Structures of dATP and 5-NITP, a prototypical non-natural nucleotide that is selectively and efficiently incorporated opposite an abasic site. (C) Strategy for using ‘clickable’ nucleotides to monitor TLS. (D) Synthesis of 3-Eth-5-NITP using the following reagents and conditions: (a) I2, KOH, DMF (b) i. NaH, 1-α-chloro-3,5-di-(O-p-toluoyl)-2-deoxy-d-ribose, anhydrous ACN, RT, 16 h; ii. NaOMe, MeOH, pH > 12, RT, 16 h; (c) Pd(PPh3)2Cl2, CuI, triethylamine, trimethylsilylacetylene, anhydrous THF, RT, 3 h; (d) 1 M TBAF, THF, RT, 3 h; (e) i. POCl3, Proton Sponge®, trimethylphosphate, 0°C; ii. Tributylammonium pyrophosphate, DMF, tributylamine, RT, 15 min; iii. 1 M TEAB, RT, 2 h.
Figure 2.
Figure 2.
Validation that non-natural nucleotides are substrates during TLS. (A) DNA substrate used for kinetic analysis. (B) Denaturing gel electrophoresis image comparing the incorporation of 5-NITP (left) and 3-Eth-5-NITP (right) opposite an abasic site and templating bases (A, C, G and T). (C) Representative time courses for the incorporation of 3-Eth-5-NITP opposite an abasic site performed using 1 nM of the exonuclease-deficient bacteriophage T4 DNA polymerase, 500 nM 13/20AP DNA substrate, and 10 mM Mg2+ in a reaction buffer with the following 3-Eth-5-NITP concentrations: 0.10 (closed circle), 0.25 (open circle), 0.50 (open square), 1.0 (closed square), 2.0 (open triangle), or 4.0 µM (closed triangle) (D) Michaelis–Menten plot for the incorporation of 3-Eth-5-NITP opposite an abasic site yielded the following kinetic parameters: kcat = 3.5 ± 0.3 s−1, Km = 0.20 ± 0.07 µM, kcat/Km = 1.75*107 M−1 s−1. (E) Experimental protocol used to evaluate the chain termination capabilities of the non-natural nucleotides used in this study. (F) Gel electrophoresis data evaluating incorporation and extension beyond an abasic site. Experiments were performed using single turnover conditions by mixing 500 nM DNA polymerase and 250 nM DNA with nucleotide and Mg2+. After four half-lives, an aliquot of the reaction was quenched with 350 mM EDTA to validate nucleotide insertion opposite the lesion. At this time, 500 µM dGTP was added to initiate elongation. As indicated, dATP can be incorporated opposite the DNA lesion and extended. While 5-NITP and 3-Eth-5-NITP are incorporated opposite the DNA lesion, they are not extended and thus behave as chain terminators.
Figure 3.
Figure 3.
(A) Denaturing gel electrophoresis validates the ability of 3-Eth-5-NITP to react with an azide-containing fluorophore. Lane 1 shows the ‘clicking’ reaction of 3-Eth-5-NITP incorporated opposite abasic-containing DNA. The upper band represents ‘clicked’ DNA containing 3-Eth-5-NIMP while the lower band represents unreacted AlexaFluor488-azide. Lane 2 shows the presence of a single fluorogenic species in ‘clicked’ reactions of DNA and 3-Eth-5-NITP without DNA polymerase. Lane 3 shows AlexaFluor488-azide alone. (B) Autoradiogram of ‘clicked’ DNA. Lane 1 represents radiolabeled primer (13-mer) in the absence of polymerase and nucleotide substrate. Lane 2 represents the incorporation of 3-Eth-5-NITP opposite an abasic site. Lane 3 represents the ‘clicking’ reaction of DNA containing 3-Eth-5-NIMP opposite an abasic site. Lane 4 represents radiolabeled primer (13-mer) in the absence of polymerase and nucleotide substrate. Lane 5 represents the incorporation of 5-NITP opposite an abasic site. Lane 6 shows the ‘clicking’ reaction of DNA containing 5-NIMP opposite an abasic site. (C) Gel electrophoresis data showing the efficiency of 3-Eth-5-NITP incorporation opposite damaged DNA. Assays were performed using 40 nM bacteriophage T4 exo− DNA polymerase, 2 µM 13/20AP-mer DNA substrate in a buffer containing 10 mM Mg2+ and 10 µM 3-Eth-5-NITP in the presence of the following concentrations of dNTPs: 0 µM (lane 1), 50 µM (lane 2), 100 µM (lane 3), 250 µM (lane 4) and 500 µM (lane 5). (D) Denaturing gel electrophoresis validating the selectivity of 3-Eth-5-NITP for replicating damaged DNA. Assays were performed using 40 nM bacteriophage T4 exo− DNA polymerase with the following conditions: 2 µM 13/20A-mer DNA substrate, 100 µM 3-Eth-5-NITP without dTTP (lane 1), 2 µM 13/20A-mer DNA substrate, 100 µM 3-Eth-5-NITP with 10 µM dTTP (lane 2), 2 µM 13/20AP-mer DNA substrate, 100 µM 3-Eth-5-NITP without dTTP (lane 3), or 2 µM 13/20AP-mer DNA substrate, 100 µM 3-Eth-5-NITP with 10 µM dTTP (lane 4).
Figure 4.
Figure 4.
(A) DNA substrate used to analyze the selective incorporation of 3-Eth-5-NITP opposite an abasic site. (B) Denaturing gel electrophoresis demonstrating the ability of the bacteriophage T4 DNA polymerase to bypass an abasic site. Reactions were performed by pre-incubating 500 nM bacteriophage T4 exo− DNA polymerase with 250 nM 13/28AP-mer and initiating the reaction with 500 µM dNTPs. Although there is slight pausing at the lesion (position 22), the polymerase can completely elongate the DNA. (C) Denaturing gel electrophoresis validates the chain termination capabilities of 3-Eth-5-NITP during TLS. Reactions were performed by pre-incubating 500 nM bacteriophage T4 exo− DNA polymerase with 250 nM 13/28AP-mer and initiating the reaction with 500 µM dNTPs and 10 µM 3-Eth-5-NITP. Note that DNA synthesis is terminated specifically at the DNA lesion (position 22).
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References

    1. Hanawalt PC. Paradigms for the three rs: DNA replication, recombination, and repair. Mol. Cell. 2007;28:702–707. - PubMed
    1. Pages V, Fuchs RP. How DNA lesions are turned into mutations within cells? Oncogene. 2002;21:8957–8966. - PubMed
    1. Huffman JL, Sundheim O, Tainer JA. DNA base damage recognition and removal: new twists and grooves. Mutat. Res. 2005;577:55–76. - PubMed
    1. Atamna H, Cheung I, Ames BN. A method for detecting abasic sites in living cells: age-dependent changes in base excision repair. Proc. Natl Acad. Sci. USA. 2000;97:686–691. - PMC - PubMed
    1. Nakamura J, Swenberg JA. Endogenous apurinic/apyrimidinic sites in genomic DNA of mammalian tissues. Cancer Res. 1999;59:2522–2526. - PubMed

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