<i>O</i>⁶‑Alkylguanine Postlesion DNA Synthesis Is Correct with the Right Complement of Hydrogen Bonding

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 <i>Sulfolobus solfataricus</i>, to perform PLS beyond the pro-mutagenic DNA adducts <i>O</i>⁶-benzylguanine (<i>O</i>⁶-BnG) and <i>O</i>⁶-methylguanine (<i>O</i>⁶-MeG). Here, <i>O</i>⁶-BnG and <i>O</i>⁶-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 <i>O</i>⁶-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:<i>O</i>⁶-MeG and Benzi:<i>O</i>⁶-BnG pairs, respectively. Correct extension was observed when <i>O</i>⁶-BnG and <i>O</i>⁶-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 <i>O</i>⁶-BnG and <i>O</i>⁶-MeG. Interestingly, Benzi was extended with high fidelity by Dpo4 when it was paired opposite <i>O</i>⁶-BnG and <i>O</i>⁶-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 <i>O</i>⁶-alkylguanine PLS

Tolerance of Base Pair Size and Shape in Postlesion DNA Synthesis

The influence of base pair size and shape on the fidelity of DNA polymerase-mediated extension past lesion-containing mispairs was examined. Primer extension analysis was performed with synthetic nucleosides paired opposite the pro-mutagenic DNA lesion <i>O</i>⁶-benzylguanine (<i>O</i>⁶-BnG). These data indicate that the error-prone DNA polymerase IV (Dpo4) inefficiently extended past the larger Peri:<i>O</i>⁶-BnG base pair, and in contrast, error-free extension was observed for the smaller BIM:<i>O</i>⁶<i>-</i>BnG base pair. Steady-state kinetic analysis revealed that Dpo4 catalytic efficiency was strongly influenced by the primer:template base pair. Compared to the C:G pair, a 1.9- and 79 000-fold reduction in Dpo4 efficiency was observed for terminal C:<i>O</i>⁶-BnG and BIM:G base pairs respectively. These results demonstrate the impact of geometrical size and shape on polymerase-mediated mispair extension

Reversible Aggregation of DNA-Decorated Gold Nanoparticles Controlled by Molecular Recognition

The programmable assembly of functional nanomaterials has been extensively addressed; however, their selective reversible assembly in response to an external stimulus has been more difficult to realize. The specificity and programmable interactions of DNA have been exploited for the rational self-assembly of DNA-conjugated nanoparticles, and here we demonstrate the sequence-controlled disaggregation of DNA-modified gold nanoparticles simply by employing two complementary oligonucleotides. Target oligonucleotides with perfectly matching sequence enabled dissociation of aggregated nanoparticles, whereas oligonucleotides differing by one nucleotide did not cause disassembly of the aggregated nanoparticles. Physical aspects of this process were characterized by UV–vis absorption, light scattering, and transmission electron microscopy. This strategy for programmed disassembly of gold nanoparticles in response to biological stimuli demonstrates a fundamentally important concept anticipated to be useful for diverse applications involving molecular recognition

The Base Pairing Partner Modulates Alkylguanine Alkyltransferase

<i>O</i>⁶-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 <i>O</i>⁶-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 <i>KRAS</i> gene with complementary sequences containing each canonical DNA base. The rate of <i>O</i>⁶-MeG repair decreased 2-fold when <i>O</i>⁶-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 <i>O</i>⁶-MeG adopting a <i>syn</i> 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

Torsional Constraints of DNA Substrates Impact Cas9 Cleavage

To examine the effect of the torsional constraints imposed on DNA substrates on Cas9 cleavage, we prepared constrained DNA substrates using a DNA origami frame. By fixing the dsDNA at the connectors of the DNA frame, we created torsionally constrained or relaxed substrates. We quantified the cleavage of constrained and relaxed substrates by Cas9 with qPCR. Moreover, we observed the Cas9/sgRNA complex bound to the DNA substrates and characterized the dissociation of the complex with high-speed atomic force microscopy. The results revealed that the constrained nontarget strand reduced the cleavage efficiency of Cas9 drastically, whereas torsional constraints on the target strand had little effect on the cleavage. The present study suggests that highly ordered and constrained DNA structures could be obstacles for Cas9 and additionally provides insights in Cas9 dissociation at a single molecule level

Nucleotides with Altered Hydrogen Bonding Capacities Impede Human DNA Polymerase η by Reducing Synthesis in the Presence of the Major Cisplatin DNA Adduct

Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template

Screening for DNA Alkylation Mono and Cross-Linked Adducts with a Comprehensive LC-MS³ Adductomic Approach

A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated and unknown DNA adducts induced by DNA alkylating agents in biological samples. Two new features were added to a previously developed approach to significantly broaden its scope, versatility, and selectivity. First, the neutral loss of a base (guanine, adenine, thymine, or cytosine) was added to the original methodology’s neutral loss of the 2′-deoxyribose moiety to allow for the detection of all DNA base adducts. Second, targeted detection of anticipated DNA adducts based on the reactivity of the DNA alkylating agent was demonstrated by inclusion of an ion mass list for data dependent triggering of MS² fragmentation events and subsequent MS³ fragmentation. Additionally, untargeted screening of the samples, based on triggering of an MS² fragmentation event for the most intense ions of the full scan, was included for detecting unknown DNA adducts. The approach was tested by screening for DNA mono and cross-linked adducts in purified DNA and in DNA extracted from cells treated with PR104A, an experimental DNA alkylating nitrogen mustard prodrug currently under investigation for the treatment of leukemia. The results revealed the ability of this new DNA adductomic approach to detect anticipated and unknown PR104A-induced mono and cross-linked DNA adducts in biological samples. This methodology is expected to be a powerful tool for screening for DNA adducts induced by endogenous or exogenous exposures

Quantification of Acylfulvene– and Illudin S–DNA Adducts in Cells with Variable Bioactivation Capacities

Illudin S and its semisynthetic analogue acylfulvene (AF) are structurally similar but elicit different biological responses. AF is a bioreductive alkylating anticancer agent with a favorable therapeutic index, while illudin S is in general highly toxic. AF toxicity is dependent on the reductase enzyme prostaglandin reductase 1 (PTGR1) for activation to a cytotoxic reactive intermediate. While illudin S can be metabolized by PTGR1, available data suggest that its toxicity does not correspond with PTGR1 function. The goal of this study was to understand how drug cytotoxicity relates to cellular bioactivation capacity and the identity and quantity of AF– or illudin S–DNA adducts. The strategy involved identification of novel illudin S–DNA adducts and their quantitation in a newly engineered SW-480 colon cancer cell line that stably overexpresses PTGR1 (PTGR1-480). These data were compared with cytotoxicity data for both compounds in PTGR1-480 versus normal SW-480 cells, demonstrating that AF forms more DNA adducts and is more cytotoxic in cells with higher levels of PTGR1, whereas illudin S cytotoxicity and adduct formation are not influenced by PTGR1 levels. Results are discussed in the context of an overall model for how changes in relative propensities of these compounds to undergo cellular processes, such as bioactivation, contributes to DNA damage, and cytotoxicity

In-Gene Quantification of <i>O</i>⁶‑Methylguanine with Elongated Nucleoside Analogues on Gold Nanoprobes

Exposure of DNA to chemicals can result in the formation of DNA adducts, a molecular initiating event in genotoxin-induced carcinogenesis. <i>O</i>⁶-Methylguanine (<i>O</i>⁶-MeG) is a highly mutagenic DNA adduct that forms in human genomic DNA upon reaction with methylating agents of dietary, environmental, or endogenous origin. In this work, we report the design and synthesis of novel non-natural nucleoside analogues 1′-β-[1-naphtho­[2,3-<i>d</i>]­imidazol-2­(3<i>H</i>)-one)]-2′-deoxy-d-ribofuranose and 1′-β-[1-naphtho­[2,3-<i>d</i>]­imidazole]-2′-deoxy-d-ribofuranose and their use for quantifying <i>O</i>⁶-MeG within mutational hotspots of the human KRAS gene. The novel nucleoside analogues were incorporated into oligonucleotides conjugated to gold nanoparticles to comprise a DNA hybridization probe system for detecting <i>O</i>⁶-MeG in a sequence-specific manner on the basis of colorimetric readout of the nanoparticles. The concept described herein is unique in utilizing new nucleoside analogues with elongated hydrophobic surfaces to successfully measure in-gene abundance of <i>O</i>⁶-MeG in mixtures with competing unmodified DNA

DNA Adduct Profiles Predict in Vitro Cell Viability after Treatment with the Experimental Anticancer Prodrug PR104A

PR104A is an experimental DNA-alkylating hypoxia-activated prodrug that can also be activated in an oxygen-independent manner by the two-electron aldo-keto reductase 1C3. Nitroreduction leads to the formation of cytotoxic hydroxylamine (PR104H) and amine (PR104M) metabolites, which induce DNA mono and cross-linked adducts in cells. PR104A-derived DNA adducts can be utilized as drug-specific biomarkers of efficacy and as a mechanistic tool to elucidate the cellular and molecular effects of PR104A. Toward this goal, a mass spectrometric bioanalysis approach based on a stable isotope-labeled adduct mixture (SILAM) and selected reaction monitoring (SRM) data acquisition for relative quantitation of PR104A-derived DNA adducts in cells was developed. Use of this SILAM-based approach supported simultaneous relative quantitation of 33 PR104A-derived DNA adducts in the same sample, which allowed testing of the hypothesis that the enhanced cytotoxicity, observed by preconditioning cells with the transcription-activating isothiocyanate sulforaphane, is induced by an increased level of DNA adducts induced by PR104H and PR104M, but not PR104A. By applying the new SILAM-SRM approach, we found a 2.4-fold increase in the level of DNA adducts induced by PR104H and PR104M in HT-29 cells preconditioned with sulforaphane and a corresponding 2.6-fold increase in cytotoxicity. These results suggest that DNA adduct levels correlate with drug potency and underly the possibility of monitoring PR104A-derived DNA adducts as biomarkers of efficacy