Effects of Chemopreventive Natural Compounds on the Accuracy of 8-oxo-7,8-dihydro-2′-deoxyguanosine Translesion Synthesis

Translesion synthesis is a DNA damage tolerance mechanism that relies on a series of specialized DNA polymerases able to bypass a lesion on a DNA template strand during replication or post-repair synthesis. Specialized translesion synthesis DNA polymerases pursue replication by inserting a base opposite to this lesion, correctly or incorrectly depending on the lesion nature, involved DNA polymerase(s), sequence context, and still unknown factors. To measure the correct or mutagenic outcome of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by translesion synthesis, a primer-extension assay was performed in vitro on a template DNA bearing this lesion in the presence of nuclear proteins extracted from human intestinal epithelial cells (FHs 74 Int cell line); the reaction products were analyzed by both denaturing capillary electrophoresis (to measure the yield of translesion elongation) and pyrosequencing (to determine the identity of the nucleotide inserted in front of the lesion). The influence of 14 natural polyphenols on the correct or mutagenic outcome of translesion synthesis through 8-oxo-7,8-dihydro-2′-deoxyguanosine was then evaluated in 2 experimental conditions by adding the polyphenol either (i) to the reaction mix during the primer extension assay; or (ii) to the culture medium, 24 h before cell harvest and nuclear proteins extraction. Most of the tested polyphenols significantly influenced the outcome of translesion synthesis, either through an error-free (apigenin, baicalein, sakuranetin, and myricetin) or a mutagenic pathway (epicatechin, chalcone, genistein, magnolol, and honokiol).

An allele-specific primer extension assay to quantify the proportion of B.1.1.7-specific SARS-CoV-2 RNA in wastewater

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives globally to date. Rapid accumulation of co-occurring mutations has led to the emergence of viral variants which appear to be more transmissible, virulent, or both. Variants of concern (VOC) now include those belonging to the B.1.1.7, B.1.351, and P.1 lineages. Early detection of VOC and the ability to retrospectively follow their respective, longitudinal prevalence in communities is wanting. Wastewater-based epidemiology (WBE) allows tracking of disease prevalence in the general population using RT-qPCR to detect viral fragments, but ongoing longitudinal studies have yet to differentiate between these variants. Here, we describe and validate a primer extension strategy to amplify and distinguish B.1.1.7-specific, from non-B.1.1.7 alleles by combining new forward, and existing CDC 2019-nCoV_N1 qPCR probes and reverse primers. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost with the goal of providing early detection of increasing B.1.1.7 transmission in a community prior to identification through clinical testing and confirmation via secondary screening strategies. As such, this assay can provide public health units with an additional and much needed metric to be able to rapidly triangulate B.1.1.7 prevalence.

Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants

ABSTRACT The present study aimed to increase the processivity of Sulfolobus solfataricus DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4. Furthermore, the A181D mutant showed lower fidelity and higher nucleotide incorporation efficiency (4.74 × 10−4 s−1 μM−1) than E63K and I248Y mutants. When tasked with bypassing damage, the A181D mutant exhibited a 3.81-fold and 2.62-fold higher catalytic efficiency (k cat/Km ) at incorporating dCTP and dATP, respectively, than wild-type Dpo4. It also showed a 55% and 91.5% higher catalytic efficiency when moving beyond the damaged 8-oxoG:C and 8-oxoG:A base pairs, respectively, compared to wild-type Dpo4. Protein engineering and bioinformatics methods can effectively increase the processivity and translesion synthesis ability of Dpo4. IMPORTANCE DNA polymerases with poor fidelity can be exploited to store data and record changes in response to the intracellular environment. Sulfolobus solfataricus Dpo4 is such an enzyme, although its use is hindered by its low processivity. In this work, we used a bioinformatics and protein engineering approach to generate Dpo4 mutants with improved processivity. We identified the Dpo4 thumb domain as the most relevant in controlling processivity.