Biology subjects

Raeder, S. B.

Publications and source records attributed to Raeder, S. B..

3 recordsLinked to original sources

bioRxiv2025

The RecBCD complex interacts directly with the DNA sliding clamp in Escherichia coli

DNA sliding clamps are essential coordinators of genome replication and maintenance across all domains of life and serve as platforms for recruiting diverse binding partners. The bacterial DNA sliding clamp, {beta}-clamp, functions analogous to the eukaryotic proliferating cell nuclear antigen (PCNA), yet its full interactome remains incompletely characterized. Here, we identify and characterize a previously unrecognized interaction between {beta}-clamp and the DNA repair helicase-nuclease complex RecBCD, specifically through its RecB subunit. Using bacterial two-hybrid assays, co-immunoprecipitation, and fluorescence microscopy, we show that RecB physically associates with {beta}-clamp. Nuclear magnetic resonance (NMR) spectroscopy demonstrates binding to the canonical ligand binding pocket in {beta}-clamp and identifies a clamp-binding motif within the RecB nuclease domain which targeted mutagenesis abolishes. Moreover, disrupting the interaction between RecB and {beta}-clamp compromises Escherichia coli survival following DNA damage and impairs the DNA degradation activity of RecBCD. These findings expand the known {beta}-clamp interactome and uncover a possible role for {beta}-clamp in DNA double-strand break repair, offering new insights into bacterial DNA metabolism and potential avenues for therapeutic intervention. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/671105v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aa96b9org.highwire.dtl.DTLVardef@8d80daorg.highwire.dtl.DTLVardef@6a1f0org.highwire.dtl.DTLVardef@171c1bc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Exploring the genetic landscape of ciprofloxacin-induced DNA supercompaction in Escherichia coli

DNA-damaging antibiotics like ciprofloxacin induce extensive double-strand breaks in Escherichia coli, triggering the SOS response and leading to DNA supercompaction. To uncover genes involved in this process beyond the previously identified core factors of recN and recA, we conducted a genome-wide screening using high-content imaging and machine learning-assisted image classification on nearly 4,000 E. coli strains, including the Keio collections single-gene deletion strains and additional in-house strains. Our investigation revealed novel genes contributing to supercompaction, with effects varying by genetic background. DNA supercompaction was consistently observed across eight clinical isolates from diverse bacterial species, underscoring the conservation of this cellular response. Our findings confirm RecN and RecA as primary drivers of DNA supercompaction. Additionally, we identified repair genes and novel genes that contribute to the response, especially in clinical E. coli strains. Notably, select hit gene deletions, including those for the membrane-associated proteins Hfq and YaiW, reduced RecN colocalization with the nucleoid, indicating a potential mechanism by which these genes impair supercompaction. Altogether, this work demonstrates that high-content imaging combined with automated analysis provides a powerful approach to explore population-level nucleoid dynamics and DNA damage responses, opening new avenues for understanding cellular processes and combating bacterial infections. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/663469v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@cfbdd9org.highwire.dtl.DTLVardef@b92022org.highwire.dtl.DTLVardef@1ecd826org.highwire.dtl.DTLVardef@62d6f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin exposure in Escherichia coli

Fluoroquinolones induce double-strand breaks in bacterial DNA, triggering the SOS response, a major DNA damage response that ensures the expression of repair proteins but also promotes the emergence and spread of antibiotic resistance. Fluoroquinolone resistance, particularly in Escherichia coli, is a growing global health concern. Understanding bacterial responses to these antibiotics is critical for developing preventive strategies and novel treatments to combat resistance development. This study investigates DNA morphology in E. coli following exposure to ciprofloxacin, a fluoroquinolone antibiotic. We show that ciprofloxacin induces a stepwise DNA reorganization, culminating in a highly dense nucleoid structure at midcell -- a process we term DNA supercompaction. Live cell imaging revealed that RecN, a structural maintenance of chromosomes (SMC)-like protein, is required for DNA supercompaction, and that RecNs dynamics and activity in this response depend on RecA. Additionally, RecN and RecA frequently colocalized at nucleoid-associated positions. We suggest that RecN and RecA play active roles in DNA supercompaction following severe DNA damage, that their interplay is part of a prompt universal survival response to DNA double-strand breaks in E. coli, and that the extent of the compaction response depends on the number of double-strand breaks. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/623168v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b948e3org.highwire.dtl.DTLVardef@607014org.highwire.dtl.DTLVardef@15e4ceforg.highwire.dtl.DTLVardef@d1b1c_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

microbiology↗