Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Poster Abstracts

14-POS Board 14 BIOCHEMICAL CHARACTERIZATION OF ENGINEERED RECA OLIGOMERS AND THEIR ROLE IN THE BACTERIAL SOS RESPONSE Allen Li 1,3 ; Michael B Cory 1 ; Christina M Hurley 1 ; Rahul M Kohli 1,2 ; 1 University of Pennsylvania, Biochemistry and Biophysics, Philadelphia, PA, USA The rapid acquisition and development of antibiotic resistance in bacteria poses a critical obstacle. While a variety of mechanisms promote antibiotic resistance, one core mechanism is the bacterial SOS response to DNA damage. This system controls key processes in bacterial survival following DNA damage, including biofilm formation, but most importantly, expression of error-prone translesion DNA polymerases. The SOS response is controlled by the interaction between two key proteins, LexA and RecA. Without DNA damage, LexA represses the expression of SOS genes. Upon DNA damage, single-stranded DNA (ssDNA) segments are generated, followed by RecA oligomerization on the ssDNA and formation of extended nucleoprotein filaments. LexA can bind to these filaments, forming the ‘SOS signal complex’, and LexA autoproteolysis then relieves repression of SOS genes. Given the nature of the LexA RecA interaction, studies to understand the molecular basis for complex formation have been hampered by the fact that any mutations in a single RecA monomer propagate throughout the filament. Previous models of the SOS signal complex have suggested that two to seven RecA monomers are required for the LexA-RecA interaction. Our work, by engineering covalently linked RecA oligomers of controlled lengths, has offered us the opportunity to identify the minimal quantity of RecA units needed for the SOS response. Furthermore, these engineered oligomers have allowed us to circumvent mutation propagation. We investigated specific residues in specific protomers of our engineered RecA oligomers and introduced mutations to probe their role in the SOS response. Our results demonstrated that point mutations in wild-type RecA can lead to a complete knockout of activity, while mutation in specific protomers of the engineered RecA can be tolerated. Ultimately, our engineered RecA oligomers lay the foundation for detailed characterization of the SOS signal complex and insights into the associated central bacterial survival mechanism. 2 University of Pennsylvania, Medicine, Philadelphia, PA, USA 3 University of Pennsylvania, Chemistry, Philadelphia, PA, USA

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