Biology subjects

Doetsch, P. W.

Publications and source records attributed to Doetsch, P. W..

4 recordsLinked to original sources

bioRxiv2026

Redox stress agents strongly enhance mutagenesis during horizontal gene transfer in bacteria and leave distinct mutational and metabolic footprints

Redox stress induces DNA mutations that contribute to chronic conditions affecting human health and to the emergence of antibiotic resistance. Yet, the impact of redox stress-induced mutagenesis remains difficult to decipher because redox agents are diverse and produce hard-to-detect mutational outcomes. Single-stranded DNA (ssDNA) provides a useful tool for studying mutagenic effects of redox agents, as it is particularly susceptible to damage and cannot be repaired by most DNA repair pathways. Here, we established a protocol to investigate redox stress-induced mutagenesis based on the Escherichia coli conjugative ssDNA that is transferred from donor to recipient cells. Using the environmentally relevant redox agents, potassium bromate and hydrogen peroxide, we show that the F episome is remarkably sensitive to weak mutagens during conjugation, enabling the detection of significant differences in mutational spectra induced by these agents. We support our findings with metabolomic analysis, which reveals agent-specific responses in E. coli. We compare these results with those obtained using a yeast ssDNA reporter and conclude that redox-induced mutagenesis depends, among other factors, on the metabolic context of the analysed system. These findings have important implications because the high sensitivity of conjugation-associated ssDNA to environmental mutagens may contribute to the evolution of antibiotic resistance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/730102v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@16a2a1eorg.highwire.dtl.DTLVardef@f329bcorg.highwire.dtl.DTLVardef@38fd4eorg.highwire.dtl.DTLVardef@17e4c9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Structural and Computational Analysis of Pseudomonas Aeruginosa DNA Gyrase Reveals Molecular Characteristics That May Contribute to Ciprofloxacin Resistance

Pseudomonas aeruginosa is considered a priority pathogen by the World Health Organization due to its resistance to antibiotics. Isolates resistant to ciprofloxacin (CPFX), a bactericide commonly used against P. aeruginosa, usually carry the mutations T83I or D87N in the GyrA subunit of the DNA gyrase. Yet, the molecular mechanisms by which these mutations confer CPFX-resistance to P. aeruginosa are unknown. Here we solved the crystal structure of the P. aeruginosa gyrase catalytic cleavage core and used it to carry out molecular dynamic (MD) simulations of CPFX-gyrase binding in the wild-type as well as the T83I and the D87N mutant systems. Our results show that DNA plays the most prominent role in maintaining the CPFX-bound conformation, with no appreciable contributions from Thr83 or Asp87. Interestingly, we found a solvent cavity adjacent to these residues that may provide CPFX access to the active site. Interaction energy analysis using Umbrella Sampling indicates that Thr83 and Asp87 may influence CPFX trajectory during binding. In the mutant systems, the attractive potential decreases, which may hinder CPFX accessing the binding site. These results shed light on P. aeruginosa resistance to CPFX and may help provide a methodology to identify new therapeutic agents to target fluoroquinolone resistant bacteria. Graphical abstract caption O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/695517v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1c203f0org.highwire.dtl.DTLVardef@90aa78org.highwire.dtl.DTLVardef@1a7d286org.highwire.dtl.DTLVardef@ee8c7a_HPS_FORMAT_FIGEXP M_FIG C_FIG Molecular dynamic positioning of ciprofloxacin (CPFX) from the cavity site (green) to the bound inhibition site (magenta) suggests that mutations T83I and D87N, conferring CPFX resistance to the P. aeruginosa gyrase, may have an influence on the ability of CPFX to access the binding site. Protein is shown in cyan, DNA backbone in orange, and DNA bases in gradient from cyan to blue.

molecular biology↗

bioRxiv2022

A Saccharomyces cerevisiae Model for Overexpression of Ntg1 a Base Excision DNA Repair Protein Reveals Novel Genetic Interactions

The Base Excision Repair (BER) pathway repairs oxidative DNA damage, a common and detrimental form of damage to the genome. Although biochemical steps in BER have been well define, little is understood about how the pathway is regulated. Such regulation is critical, as cells must respond rapidly to DNA damage while avoiding aberrant activation of repair proteins that can produce DNA damage as intermediates in the repair pathway. Indeed, overexpression of the human BER protein, NTHL1, a DNA N-glycosylase, can cause genomic instability and early cellular hallmarks of cancer. We developed a Saccharomyces cerevisiae model to explore how overexpression of NTHL1 may impair cellular function. Overexpression of Ntg1, the budding yeast orthologue of NTHL1, impairs cell growth. To dissect mechanisms underlying this growth defect, we overexpressed either wild-type Ntg1 or a catalytically inactive variant of Ntg1 (ntg1catdead). Consistent with results obtained for NTHL1, both variants of Ntg1 impair cell growth, but only the wild-type protein causes accumulation of double-strand breaks and chromosome loss. We screened a panel of DNA repair mutants for resistance/sensitivity to overexpression of wild-type Ntg1 or ntg1catdead. This analysis identified several cellular pathways that protect cells from Ntg1-induced damage, providing insight into interplay between DNA repair pathways. Finally, we identified a link to sumoylation and probed how this post-translational modification could contribute to regulation of Ntg1 function. This study describes a budding yeast system to understand how cells regulate and respond to dysregulation of the BER pathway. Take AwayO_LIOverexpression of a base excision DNA repair protein impairs cell growth C_LIO_LIOverexpression of a base excision DNA repair protein can cause DNA damage C_LIO_LIMultiple mechanisms cause DNA damage from overexpression of a repair protein C_LIO_LIDNA repair pathways functionally interact to protect cells from DNA damage C_LIO_LIPrecise regulation of the activity of DNA repair proteins is critical C_LI

genetics↗

bioRxiv2020

Replication stress and FOXM1 drive radiation induced genomic instability and cell transformation

In contrast to the vast majority of research that has focused on the immediate effects of ionizing radiation, this work concentrates on the molecular mechanism driving delayed effects that emerge in the progeny of the exposed cells. We employed functional protein arrays to identify molecular changes induced in a human bronchial epithelial cell line (HBEC3-KT) and osteosarcoma cell line (U2OS) and evaluated their impact on outcomes associated with radiation induced genomic instability (RIGI) at day 5 and 7 post-exposure to a 2Gy X-ray dose, which revealed replication stress in the context of increased FOXM1 expression. Irradiated cells had reduced DNA replication rate detected by the DNA fiber assay and increased DNA resection detected by RPA foci and phosphorylation. Irradiated cells increased utilization of homologous recombination-dependent repair detected by a gene conversion assay and DNA damage at mitosis reflected by RPA positive chromosomal bridges, micronuclei formation and 53BP1 positive bodies in G1, all known outcomes of replication stress. Interference with the function of FOXM1, a transcription factor widely expressed in cancer, employing an aptamer, decreased radiation-induced micronuclei formation and cell transformation while plasmid-driven overexpression of FOXM1b was sufficient to induce replication stress, micronuclei formation and cell transformation.

cell biology↗