Kaminski, A. M. | bioRxiv Science

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↗

bioRxiv2025

End Processing in NHEJ by Polymerase {lambda} and PNKP is coordinated during short-range synapsis

Non-homologous end joining (NHEJ) is a major pathway of DNA double strand break (DSB) repair, capable of directly joining both damaged strands of DNA through the coordinated activities of repair factors that detect the termini, physically bridge them together, and perform the chemistry necessary to complete repair. NHEJ is capable of repairing a variety of damaged DNA, employing various accessory end-processing factors to resolve chemically blocked ends, trim overhangs, and fill gaps in order to achieve directly ligatable DNA ends. To investigate the molecular mechanisms underlying end-processing, we determined the cryo-EM structure of the NHEJ specific polymerase Pol {lambda} bound to the short-range synaptic complex, uncovering the mode of its recruitment to the complex as well as a putative model for its activity. Furthermore, the coordinated end-processing activities of the short-range (SR) synaptic complex simultaneously bound by both Pol {lambda} and PNKP, another accessory factor, demonstrates the ability of NHEJ to form large, multifunctional repair complexes capable of processing a variety of different DNA end structures to effect repair.

biophysics↗