Abstact

The Escherichia coli yqgF gene product is essential for bacterial growth and to confer resistance to multiple antimicrobial agents. Furthermore, additional evidence suggests that YqgF, a RuvC family protein, is required for DNA damage repair, yet the mechanism underlying its action remains elusive. To address this knowledge gap, we conducted structural and biochemical investigations on E. coli YqgF (EcYqgF). Here, we reveal that EcYqgF binds preferentially to branched DNA structures compared to single-stranded (ssDNA) and double-stranded DNA (dsDNA), and that the EcYqgF:DNA complexes formed with branched DNA species were more stable and resistant against high salt and excess of competitor DNA than those formed with dsDNA. We show that EcYqgF has a strong preference towards cleavage of branched DNA structures than dsDNA, ssDNA, and dsDNA with 5'- or 3'-ssDNA overhangs. Crucially, we found that EcYqgF has a DNA-independent, Mg2+-dependent ATPase activity that is tightly coupled to DNA cleavage. We have determined the crystal structure of EcYqgF, developed a model of ATP binding using AI-based methods, and rationalized the impact of site-directed mutants on ATP binding. Furthermore, we discovered two unusual ATPase-defective EcYqgF variants, proficient in ATP-binding but not hydrolysis, which display a modest increase in the DNA-binding affinity, yet are devoid of endonucleolytic activity, thus revealing a previously unappreciated property of YqgF endonucleases. Collectively, our results suggest that despite its overall structural similarity to the well-studied resolvase, RuvC, EcYqgF is functionally distinct. Importantly, the EcYqgF dual activity that couples ATP hydrolysis to endonuclease activity is absent in RuvC.