Variable inhibition of unwinding rates of DNA catalyzed by the SARS-Cov-2 (COV19) helicase nsp13 by structurally distinct single DNA lesions.

The SARS 2 (Covid 19) helicase nsp13 plays a critically important role in the replication of the Corona virus by unwinding double-stranded RNA (and DNA) with a 5 prime to 3 prime strand polarity. Here we explored the impact of single, structurally defined covalent DNA lesions on the helicase activity of nsp13 in aqueous solutions, The objectives were to derive mechanistic insights into the relationships between the structures of DNA lesions, the DNA distortions that they engender, and the inhibition of helicase activity. The lesions included two bulky stereoisomeric N2-guanine adducts derived from the reactions of benzo[a]pyrene diol epoxide with DNA. The trans-adduct assumes a minor groove conformation, while the cis-product adopts a base-displaced intercalated conformation. The non-bulky DNA lesions included the intra-strand cross-linked thymine dimers, the cis-syn-cyclobutane pyrimidine dimer, and the pyrimidine (6–4) pyrimidone photoproduct. All four lesions strongly inhibit the helicase activity of nsp13, The UV photolesions feature a 2 - 5-fold smaller inhibition of the nsp13 unwinding activity than the bulky DNA adducts, and the kinetics of these two pairs of DNA lesions are also different. The connections between the structural features of these four DNA lesions and their impact on nsp13 unwinding efficiencies are discussed.

A Kinetic Analysis of DNA-Deoxy Guanine Adducts in the Nasal Epithelium Produced by Inhaled Formaldehyde in Rats—Assessing Contributions to Adduct Production From Both Endogenous and Exogenous Sources of Formaldehyde

Although formaldehyde is a normal constituent of tissues, lifetime inhalation exposures at 6 h/day, 5 days/week at concentrations ≥6 ppm caused a nonlinear increase in nasal tumors in rats with incidence reaching close to 50% at 15 ppm. Studies with heavy isotope labeled [13CD2]-formaldehyde permit quantification of both the mass-labeled exogenous and endogenous DNA-formaldehyde reaction products. An existing pharmacokinetic model developed initially to describe 14C-DNA-protein crosslinks (DPX) provided a template for describing the time course of mass-labeled adducts. Published datasets included both DPX and N2-HO13CD2-dG adducts measured after a single 6-h exposure to 0.7, 2, 6, 9, 10, or 15 ppm formaldehyde, after multi-day exposures to 2 ppm for 6 h/day, 7 days/week with interim sacrifices up to 28 days, and after 28-day exposures for 6 h/day, 7 days/week to 0.3, 0.03, or 0.001 ppm. The existing kinetic model overpredicted endogenous adducts in the nasal epithelium after 1-day [13CD2]-formaldehyde exposure, requiring adjustment of parameters for rates of tissue metabolism and background formaldehyde. After refining tissue formaldehyde parameters, we fit the model to both forms of adducts by varying key parameters and optimizing against all 3 studies. Fitting to all these studies required 2 nonlinear pathways—one for high-exposure saturation of clearance in the nasal epithelial tissues and another for extracellular clearance that restricts uptake into the epithelial tissue for inhaled concentrations below 0.7 ppm. This refined pharmacokinetic model for endogenous and exogenous formaldehyde acetal adducts can assist in updating biologically based dose-response models for formaldehyde carcinogenicity.

Urinary N7-(1-hydroxy-3-buten-2-yl) guanine adducts in humans: temporal stability and association with smoking

1,3-Butadiene (BD) is a known human carcinogen found in cigarette smoke, automobile exhaust, and urban air. Workers occupationally exposed to BD in the workplace have an increased incidence of leukemia and lymphoma. BD undergoes cytochrome P450-mediated metabolic activation to 3,4-epoxy-1-butene (EB), 1,2,3,4-diepoxybutane (DEB) and 1,2-dihydroxy-3,4-epoxybutane (EBD), which form covalent adducts with DNA. We have previously reported a quantitative nanoLC/ESI+-HRMS3 method for urinary N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts as a mechanism-based biomarker of BD exposure. In the present study, the method was updated to include high throughput 96-well solid phase extraction (SPE) and employed to establish urinary EB-GII biomarker stability and association with smoking. Urinary EB-GII levels were measured bimonthly for 1 year in 19 smokers to determine whether single adduct measurement provides reliable levels of EB-GII in an individual smoker. In addition, association of EB-GII with smoking was studied in 17 individuals participating in a smoking cessation program. EB-GII levels decreased 34% upon smoking cessation, indicating that it is associated with smoking status, but may also originate from sources other than exposure to cigarette smoke.

Epigenetic loss of heterozygosity of Apc and an inflammation-associated mutational signature detected in Lrig1+/--driven murine colonic adenomas

Murine colonic adenomas induced by the loss of a single copy of the tumor suppressor gene Apc in Lrig1+/- expressing progenitor cells grow rapidly, with high penetrance and tumor multiplicity. This study investigates the prevalence of intertumoral genetic heterogeneity and phenotypic variation across tumors, and attempts to identify the genomic cause of the unusual phenotype. Adult Lrig1-CreERT2/+; Apc-flox/+ mice were intraperitoneal injected with 2mg tamoxifen for 3 consecutive days which induced highly penetrant, dysplastic adenomas in the distal colon ∼100 days later. Whole tumors (n=14) from 8 mice were excised and tumor exome DNA and mRNA were sequenced. Somatic mutations present in the tumor exome DNA were compared with adjacent normal colon (n=9 tumors from 3 mice). Putative somatic mutations were called after stringent filtering using SeuratSomatic, a Genome Analysis Toolkit software module. RNA-Seq was performed on tumor mRNA (n=5 tumors from 5 mice) compared to wildtype colon (n=3). Differential gene expression was profiled using the R package DESeq2. Copy number variations and splicing defects were assessed using custom tracks on the UCSC genome browser.Adenomas resulting from the loss of Apc in Lrig1+/-- expressing colonic progenitor cells are genetically heterogeneous and hypermutated, containing ∼25-30 high-quality somatic mutations per megabase. A loss of heterozygosity of Apc was not observed in the tumor genomes, however evidence of an epigenetic loss of heterozygosity was readily apparent in the tumor transcriptome. The tumors display a strong bias toward G: C > A: T point mutations, which are a signature of guanine adducts, associated with tobacco tar and H. pylori infections. Putative tumor-driving mutations were detected and thousands of differentially expressed genes were identified including several UDP glucuronosyltransferases. Abnormal splicing patterns characterized by a loss of intron retention were detected in several RNA-binding genes throughout the tumor transcriptome. The widespread defects in gene expression, genomic stability, and splicing patterns implies that an early epigenetic loss of Apc in Lrig1+/--expressing progenitor cells causes a rapid formation of guanine adducts and a corresponding accumulation of mutation C>A point mutations. This study demonstrates that randomly-appearing oncogenic mutations can become fixed into a latent genomic reservoir prior to advanced disease.