Biology subjects

Westerlund, F.

Publications and source records attributed to Westerlund, F..

3 recordsLinked to original sources

bioRxiv2025

It takes two (Ku) to repair: Mechanistic insights into the minimal NHEJ system of Mycobacterium Tuberculosis

In this study, we used a combination of structural simulations, ensemble assays and single molecule analysis to shed light on Non-Homologous End-Joining (NHEJ) in Mycobacterium Tuberculosis (Mtb), executed by the homodimeric Ku and Ligase D (LigD). We used a monomeric form of the Ku protein to confirm the necessity of homodimerization of Ku to bind DNA. We demonstrated that Mtb Ku and shows limited translocation on DNA and primarily instead stays bound at the DNA ends. The reconstitution of an active Mtb NHEJ machinery, consisting of Ku and LigD, allowed us to characterize the dynamics of each step of the assembly of the Mtb NHEJ machinery at the single molecule level, revealing competition between Ku and LigD for DNA binding. In silico investigations highlighted key residues in Mtb LigD - Ku complex formation. We conducted a mutational analysis of these residues in the polymerase and the ligase domains, respectively. This investigation demonstrated that, although formation of the NHEJ complex is preserved in LigD mutants, mutations in the polymerase domain result in reduced enzymatic activity, whereas mutations in the ligase domain led to its enhancement. Finally, we present a model for NHEJ in M. tuberculosis, where Ku must stay tightly at the DNA ends to properly recruit and regulate LigD enzymatic activity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/694593v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@9724ccorg.highwire.dtl.DTLVardef@f0dcc4org.highwire.dtl.DTLVardef@12df36dorg.highwire.dtl.DTLVardef@1c881ea_HPS_FORMAT_FIGEXP M_FIG C_FIG Mtb Ku and LigD compete for DNA binding, while interactions between Ku and the LigD polymerase (POL) and ligase (LIG) domains differentially modulate enzymatic activity. This image was generated with the assistance of ChatGPT and Gemini and subsequently edited by the authors.

biochemistry↗

bioRxiv2025

MYC modulates TOP2A diffusion to promote substrate detection and activity

Topoisomerases alleviate DNA supercoiling by cleaving and resealing DNA strands. Previously, we showed that the oncoprotein MYC recruits and stimulates topoisomerases to remove DNA entanglements generated by oncogenic transcription. Understanding this mechanism may suggest methods to inhibit MYC-driven topoisomerase activation, targeting tumor-specific transcription. Here, we demonstrate that the essential topoisomerase TOP2A in human cells exists in a dynamic equilibrium between sequestration in the nucleolus, substrate searching in transcription hubs, and active engagement on chromatin. This equilibrium is highly responsive to changes in DNA topology, allowing cells to regulate TOP2A levels. Using single molecule tracking, we show that MYC accelerates TOP2A diffusion in cells. We explain this phenotype by demonstrating that MYC limits TOP2A self-interaction in vitro, while decreasing the size of TOP2A complexes in cells. By increasing TOP2A diffusion, MYC promotes substrate binding and increases TOP2A engagement on chromatin genome-wide, revealing the mechanism underlying MYC stimulation of TOP2A activity.

cell biology↗

bioRxiv2025

A Platinum Butterfly Effect: Small Changes Turn an Anticancer Drug into a Non-toxic Metalloantibiotic with In Vivo Efficacy

Widespread resistance to all clinically used antibiotics has sparked investigations into alternative sources for novel and effective antimicrobial agents. Metal-based compounds (metalloantibiotics) have emerged as a promising class of potential antibiotics exhibiting high hit rates against critical bacterial pathogens while not displaying higher toxicity than organic compounds. Here, we describe the exploration of a novel class of non-toxic, Gram-positive acting platinum-based antibacterial agents with micro to nanomolar activity against a range of methicillin and vancomycin-resistant Staphylococcus aureus strains. Structure-activity relationship (SAR) studies revealed that modifications of the core scaffold result in reduced antibacterial activity. Mode of action studies investigations showed that lead compound Pt1 did not impair cell division, RNA, protein, or cell wall synthesis, nor did it affect membrane integrity or potential. Instead, akin to the structurally similar anticancer drug cisplatin (CisPt), Pt1 treatment resulted in reduced DNA staining, visible nucleoid compaction, and activation of DNA damage repair responses. Importantly, we could show that Pt1 is able to interact with and damage DNA directly, resulting in DNA strand breaks and fragmentation. Pt1 activity can be reduced significantly by high amounts of a hydroxyl radical scavenger. Derivative Pt8, which retained DNA-damaging activity but was less potent in terms of antibacterial activity, was not affected by the presence of radical scavengers, suggesting that Pt1 possesses a multimodal mechanism. In line with this observation, no resistance development to Pt1 was observed over the course of 36 passages. Finally, we could demonstrate the in vivo activity of Pt1, which significantly reduced the bacterial load in a murine S. aureus skin infection model. Altogether, these findings shed light on the SAR and antibacterial mode of action of a novel class of platinum metalloantibiotics, validate its in vivo efficacy, and pave the way for further exploration of platinum compounds as novel drug candidates with a highly attractive activity profile.

microbiology↗