Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize <scp>DNA</scp> Duplexes Containing <i>O</i><sup>6</sup>‐Alkylguanine Adducts

Dahlmann, Heidi A.; Berger, Florence D.; Kung, Ryan W.; Wyss, Laura A.; Gubler, Irina; McKeague, Maureen; Wetmore, Stacey D.; Sturla, Shana J.

doi:10.1002/hlca.201800066

Cited by 4 publications

(15 citation statements)

References 75 publications

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“…The predicted structure with the lowest potential energy was O 6 -MeG( syn ):Per( syn ), which precludes hydrogen bonding (Figure B,C). This observation is consistent with a previous study indicating that π-stacking interactions of these nucleobase analogues promote duplex stability . However, the next-lowest-energy structure was with Per in the anti conformation (Figure S11), thus restoring hydrogen bonding.…”

Section: Results and Discussionsupporting

confidence: 92%

“…This observation is consistent with a previous study indicating that π-stacking interactions of these nucleobase analogues promote duplex stability. 76 However, the nextlowest-energy structure was with Per in the anti conformation (Figure S11), thus restoring hydrogen bonding. Thus, whether Per is in the syn or anti conformation, for both lowest-energy conformations, O 6 -MeG is predicted to be in a syn conformation.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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The Base Pairing Partner Modulates Alkylguanine Alkyltransferase

et al. 2018

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O-Alkylguanine DNA adducts are repaired by the suicide enzyme alkylguanine alkyltransferase (AGT). AGT facilitates repair by binding DNA in the minor groove, flipping out the damaged base, and transferring the O-alkyl group to a cysteine residue in the enzyme's active site. Despite there being significant knowledge concerning the mechanism of AGT repair, there is limited insight regarding how altered interactions of the adduct with its complementary base in the DNA duplex influence its recognition and repair. In this study, the relationship of base pairing interactions and repair by human AGT (hAGT) was tested in the frequently mutated codon 12 of the KRAS gene with complementary sequences containing each canonical DNA base. The rate of O-MeG repair decreased 2-fold when O-MeG was paired with G, whereas all other canonical bases had no impact on the repair rate. We used a combination of biochemical studies, molecular modeling, and artificial nucleobases to elucidate the mechanism accounting for the 2-fold decrease. Our results suggest that the reduced rate of repair is due to O-MeG adopting a syn conformation about the glycosidic bond precluding the formation of a repair-active complex. These data provide a novel chemical basis for how direct reversion repair may be impeded through modification of the base pair partner and support the use of artificial nucleobases as tools to probe the biochemistry of damage repair processes.

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Section: Results and Discussionsupporting

confidence: 92%

Section: ■ Results and Discussionmentioning

confidence: 99%

The Base Pairing Partner Modulates Alkylguanine Alkyltransferase

et al. 2018

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“…DNA adduct-directed artificial nucleotides have been developed as a basis for interrogating damaged DNA in duplex hybridization or polymerase-mediated synthesis contexts . We have reported a variety of nucleoside analogues that stabilize damaged DNA vs undamaged DNA. − The heterocyclic imidic nucleoside analogue derived from benzimidazolinone (Benzi) and a derivative with an extended ρ-surface termed ExBenzi (Figure B) consist of a conjugated π-system, a hydrogen bond accepting carbonyl group, and an imino-based hydrogen bond donor. When placed opposite O 6 -alkylG adducts, the resulting duplexes are stabilized. − When ExBenzi was included in short oligonucleotides attached to gold nanoparticles, the presence of O 6 -alkylG adducts in target DNA strands disrupted nanoparticle aggregation and induced a color change, indicating the presence of the adduct .…”

Section: Introductionmentioning

confidence: 99%

“…We have reported a variety of nucleoside analogues that stabilize damaged DNA vs undamaged DNA. − The heterocyclic imidic nucleoside analogue derived from benzimidazolinone (Benzi) and a derivative with an extended ρ-surface termed ExBenzi (Figure B) consist of a conjugated π-system, a hydrogen bond accepting carbonyl group, and an imino-based hydrogen bond donor. When placed opposite O 6 -alkylG adducts, the resulting duplexes are stabilized. − When ExBenzi was included in short oligonucleotides attached to gold nanoparticles, the presence of O 6 -alkylG adducts in target DNA strands disrupted nanoparticle aggregation and induced a color change, indicating the presence of the adduct . These hybridization probes provide an excellent quantitative read-out, but do not involve DNA amplification and have so far been used to detect down to 138 fmol of modified DNA in 6 pmol of total DNA.…”

Section: Introductionmentioning

confidence: 99%

Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide

et al. 2020

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DNA mutations can result from replication errors due to different forms of DNA damage, including low-abundance DNA adducts induced by reactions with electrophiles. The lack of strategies to measure DNA adducts within genomic loci, however, limits our understanding of chemical mutagenesis. The use of artificial nucleotides incorporated opposite DNA adducts by engineered DNA polymerases offers a potential basis for site-specific detection of DNA adducts, but the availability of effective artificial nucleotides that insert opposite DNA adducts is extremely limited, and furthermore, there has been no report of a quantitative strategy for determining how much DNA alkylation occurs in a sequence of interest. In this work, we synthesized an artificial nucleotide triphosphate that is selectively inserted opposite O 6-carboxymethyl-guanine DNA by an engineered polymerase and is required for DNA synthesis past the adduct. We characterized the mechanism of this enzymatic process and demonstrated that the artificial nucleotide is a marker for the presence and location in the genome of O 6-carboxymethyl-guanine. Finally, we established a mass spectrometric method for quantifying the incorporated artificial nucleotide and obtained a linear relationship with the amount of O 6-carboxymethyl-guanine in the target sequence. In this work, we present a strategy to identify, locate, and quantify a mutagenic DNA adduct, advancing tools for linking DNA alkylation to mutagenesis and for detecting DNA adducts in genes as potential diagnostic biomarkers for cancer prevention.

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“…24 Dangling-end T m measurements, where the synthetic nucleoside is unpaired and overlaps with the adjacent terminal base pair, confirmed that ExBIM and ExBenzi stabilized the duplex more than Benzi and BIM and supported the hypothesis for enhanced surface area overlap with the neighboring base on the opposing strand. 24 Further, while Benzi, BIM, ExBenzi, and ExBIM are fluorescent (quantum yields Φ f = 0.35, 0.82, 0.69, and 0.44, respectively), the fluorescence of ExBIM was the most sensitive to the altered chemical environment imposed by the presence of the adduct. Thus, ExBIM showed the highest adduct selectivity.…”

Section: ■ Introductionmentioning

confidence: 99%

DNA Adduct-Directed Synthetic Nucleosides

et al. 2019

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Conspectus Chemical damage to DNA is a key initiator of adverse biological consequences due to disruption of the faithful reading of the genetic code. For example, O 6-alkylguanine (O 6-alkylG) DNA adducts are strongly miscoding during DNA replication when the damaged nucleobase is a template for polymerase-mediated translesion DNA synthesis. Thus, mutations derived from O 6-alkylG adducts can have severe adverse effects on protein translation and function and are an early event in the initiation of carcinogenesis. However, the low abundance of these adducts places significant limitations on our ability to relate their presence and biological influences with resultant mutations or disease risk. As a consequence, there is a critical need for novel tools to detect and study the biological role of alkylation adducts. Incorporating DNA bases with altered structures that are derived synthetically is a strategy that has been used widely to interrogate biological processes involving DNA. Such synthetic nucleosides have contributed to our understanding of DNA structure, DNA polymerase (Pol) and repair enzyme function, and to the expansion of the genetic alphabet. This Account describes our efforts toward creating and applying synthetic nucleosides directed at DNA adducts. We synthesized a variety of nucleosides with altered base structures that complement the altered hydrogen bonding capacity and hydrophilicity of O 6-alkylG adducts. The heterocyclic perimidinone-derived nucleoside Per was the first of such adduct-directed synthetic nucleosides; it specifically stabilized O 6-benzylguanine (O 6-BnG) in a DNA duplex. Structural variants of Per were used to determine hydrogen bonding and base-stacking contributions to DNA duplex stability in templates containing O 6-BnG as well as O 6-methylguanine (O 6-MeG) adducts. We created synthetic probes able to stabilize damaged over undamaged templates and established how altered hydrogen bonding or base-stacking properties impact DNA duplex stability as a function of adduct structures. This knowledge was then applied to devise a hybridization-based detection strategy involving gold nanoparticles that distinguish damaged from undamaged DNA by colorimetric changes. Furthermore, synthetic nucleosides were used as mechanistic tools to understand chemical determinants such as hydrogen bonding, π-stacking, and size and shape deviations that impact the efficiency and fidelity of DNA adduct bypass by DNA Pols. Finally, we reported the first example of amplifying alkylated DNA, accomplished by combining an engineered polymerase and synthetic triphosphate for which incorporation is templated by a DNA adduct. The presence of the synthetic nucleoside in amplicons could serve as a marker for the presence and location of DNA damage at low levels in DNA strands. Adduct-directed synthetic nucleosides have opened new concepts to interrogate the levels, locations, and biological influences of DNA alkylation.

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Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize DNA Duplexes Containing O⁶‐Alkylguanine Adducts

Cited by 4 publications

References 75 publications

The Base Pairing Partner Modulates Alkylguanine Alkyltransferase

The Base Pairing Partner Modulates Alkylguanine Alkyltransferase

Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide

DNA Adduct-Directed Synthetic Nucleosides

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Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize DNA Duplexes Containing O6‐Alkylguanine Adducts

Cited by 4 publications

References 75 publications

The Base Pairing Partner Modulates Alkylguanine Alkyltransferase

The Base Pairing Partner Modulates Alkylguanine Alkyltransferase

Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide

DNA Adduct-Directed Synthetic Nucleosides

Contact Info

Product

Resources

About

Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize DNA Duplexes Containing O⁶‐Alkylguanine Adducts