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bioRxiv · 10.1101/2025.08.20.671105

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

Riisnaes, I. M. M.·Raeder, S. B.·Simonsen, S.·Vikedal, K.·Backe, P. H.·Johnsen, L.·Bjoras, M.·Booth, J. A.·Kragelund, B. B.·Skarstad, K.·Helgesen, E.
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Abstract

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

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Riisnaes, I. M. M., Raeder, S. B., Simonsen, S., Vikedal, K., Backe, P. H., Johnsen, L., Bjoras, M., Booth, J. A., Kragelund, B. B., Skarstad, K., Helgesen, E.. 2025-08-20. The RecBCD complex interacts directly with the DNA sliding clamp in Escherichia coli. https://doi.org/10.1101/2025.08.20.671105

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