Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Poster Abstracts

7-POS Board 7 DEFINING THE ACTIVATING SIGNAL OF THE SOS RESPONSE – A MODULATOR OF BACTERIAL EVOLUTION. Michael B Cory 1 ; Allen Li 2 ; Rahul M Kohli 1,3 ; 1 University of Pennsylvania, Perelman School of Medicine, Biochemistry and Biophysics, Philadelphia, PA, USA 2 University of Pennsylvania, Chemistry, Philadelphia, PA, USA 3 University of Pennsylvania, Perelman School of Medicine, Infectious Diseases, Philadelphia, PA, USA Bacteria’s ability to rapidly acquire resistance-engendering mutations presents a formidable medical challenge. Understanding mechanisms that promote adaptation to antimicrobial stress could facilitate the development of strategies to preempt acquired resistance. One such pro mutagenic bacterial pathway is the SOS response – a highly conserved genetic network that mediates DNA damage repair and tolerance. Activation of the SOS response is dependent on the interaction between the bacterial proteins RecA and LexA. RecA acts as a DNA damage sensor by forming active oligomeric filaments (RecA*) along single-stranded DNA in an ATP dependent manner. RecA* can then interact with the repressor LexA, leading to its eventual degradation. Formation of the RecA*-LexA complex, termed the SOS signal complex, initiates downstream expression of SOS response genes. While understanding this complex is key to therapeutic intervention, the minimal determinants of SOS activation remain unknown, including the number of RecA monomers in RecA* that engage with LexA in the SOS complex and their requisite activation state. Here, we leveraged constrained RecA constructs as a tool to define the minimally sufficient RecA* filament for LexA engagement. By probing their capacity for filamentation, LexA binding, and induction of LexA degradation, we reveal unexpected insights into the SOS complex. In contrast with prevailing models, we found that two RecA units are sufficient for LexA cleavage, and that both DNA- and ATP-dependent activation of the filament is strictly required. Maximal activity is observed with four RecA units, suggesting auxiliary interactions may be optimal for LexA engagement. By introducing mutations into these RecA constructs, we further demonstrate that prior models based on mutations in monomeric RecA provided misleading results on the nature of the SOS complex. With this minimal system, we are now poised to investigate the precise nature of the SOS signal complex, ultimately supporting the goal of structure-guided design of potential SOS inhibitors.

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