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bioRxiv · 10.1101/2022.09.30.510415

Engineered RecA constructs reveal the minimal SOS activation complex

Cory, M. B.·Li, A.·Hurley, C. M.·Hostetler, Z. M.·Venkatesh, Y.·Jones, C. M.·Petersson, E. J.·Kohli, R. M.
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Abstract

The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacterias ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key SOS activation signal. Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that permit LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/510415v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@149d719org.highwire.dtl.DTLVardef@c8dd3corg.highwire.dtl.DTLVardef@1349098org.highwire.dtl.DTLVardef@fb29_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Cory, M. B., Li, A., Hurley, C. M., Hostetler, Z. M., Venkatesh, Y., Jones, C. M., Petersson, E. J., Kohli, R. M.. 2022-10-01. Engineered RecA constructs reveal the minimal SOS activation complex. https://doi.org/10.1101/2022.09.30.510415

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