Replication across Regioisomeric Ethylated Thymidine Lesions by Purified DNA Polymerases

Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA, and higher levels of ethylated DNA lesions were observed in smokers than in nonsmokers. In this study, we examined comprehensively how the regioisomeric <i>O</i>²-, <i>N</i>3-, and <i>O</i>⁴-ethylthymidine (<i>O</i>²-, <i>N</i>3-, and <i>O</i>⁴-EtdT, respectively) perturb DNA replication mediated by purified human DNA polymerases (hPols) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf^–) of <i>Escherichia coli</i> DNA polymerase I. Our results showed that hPol η and Kf^– could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol κ and yPol ζ differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove <i>N</i>²-substituted 2′-deoxyguanosine lesions, hPol κ was able to bypass <i>O</i>²-EtdT, though it experienced great difficulty in bypassing <i>N</i>3-EtdT and <i>O</i>⁴-EtdT. yPol ζ was only modestly blocked by <i>O</i>⁴-EtdT, but the polymerase was strongly hindered by <i>O</i>²-EtdT and <i>N</i>3-EtdT. LC–MS/MS analysis of the replication products revealed that DNA synthesis opposite <i>O</i>⁴-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of <i>O</i>²-EtdT and <i>N</i>3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol ι and Kf^–, dTMP. Thus, our results suggested that <i>O</i>²-EtdT and <i>N</i>3-EtdT may also contribute to the AT → TA and AT → GC mutations observed in cells and tissues of animals exposed to ethylating agents

<i>In-Vitro</i> Replication Studies on <i>O</i>²‑Methylthymidine and <i>O</i>⁴‑Methylthymidine

<i>O</i>²- and <i>O</i>⁴-methylthymidine (<i>O</i>²-MdT and <i>O</i>⁴-MdT) can be induced in tissues of laboratory animals exposed with <i>N</i>-methyl-<i>N</i>-nitrosourea, a known carcinogen. These two <i>O</i>-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, <i>in vitro</i> replication studies with duplex DNA substrates containing site-specifically incorporated <i>O</i>²-MdT and <i>O</i>⁴-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of <i>Escherichia coli</i> DNA polymerase I (Kf^–), human DNA polymerase κ (pol κ), and <i>Saccharomyces cerevisiae</i> DNA polymerase η (pol η). Results from steady-state kinetic measurements and LC-MS/MS analysis of primer extension products revealed that Kf^– and pol η preferentially incorporated the correct nucleotide (dAMP) opposite <i>O</i>²-MdT, while <i>O</i>⁴-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite <i>O</i>²-MdT and <i>O</i>⁴-MdT is highly promiscuous, LC-MS/MS analysis of primer extension products demonstrated that pol κ favorably incorporated the incorrect dGMP opposite both lesions. Our results underscored the limitation of the steady-state kinetic assay in determining how DNA lesions compromise DNA replication <i>in vitro</i>. In addition, the results from our study revealed that, if left unrepaired, <i>O</i>-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents

Chemical Structure and Properties of Interstrand Cross-Links Formed by Reaction of Guanine Residues with Abasic Sites in Duplex DNA

A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG–AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link “remnant” released by enzymatic digestion of DNA duplexes containing the dG–AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant <b>9b</b> composed of a 2-deoxyribose adduct attached to the exocyclic <i>N</i>²-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (<b>11b</b>). Liquid chromatography–tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards <b>9b</b> and <b>11b</b> matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG–AP cross-link, respectively. These results establish the chemical connectivity of the dG–AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG–AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG–AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication

Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate <i>in Vivo</i> Deamidation of Therapeutic Antibodies

Deamidation of therapeutic antibodies may result in decreased drug activity and undesirable changes in pharmacokinetics and immunogenicity. Therefore, it is necessary to monitor the deamidation levels [during storage] and after <i>in vivo</i> administration. Because of the complexity of <i>in vivo</i> samples, immuno-affinity capture is widely used for specific enrichment of the target antibody prior to LC–MS. However, the conventional use of bead-based methods requires large sample volumes and extensive processing steps. Furthermore, with automation difficulties and extended sample preparation time, bead-based approaches may increase artificial deamidation. To overcome these challenges, we developed an automated platform to perform tip-based affinity capture of antibodies from complex matrixes with rapid digestion and peptide elution into 96-well microtiter plates followed by LC–MS analysis. Detailed analyses showed that the new method presents high repeatability and reproducibility with both intra and inter assay CVs < 8%. Using the automated platform, we successfully quantified the levels of deamidation of a humanized monoclonal antibody in cynomolgus monkeys over a time period of 12 weeks after administration. Moreover, we found that deamidation kinetics between <i>in vivo</i> samples and samples stressed <i>in vitro</i> at neutral pH were consistent, suggesting that the <i>in vitro</i> stress test may be used as a method to predict the liability to deamidation of therapeutic antibodies <i>in vivo</i>