The crystal structure and biochemical analyses of escherichia coli yqgf illuminate its diverse functions

Abstract

The Escherichia coli yqgF gene product is essential for bacterial growth , to confer resistance to mul- tiple 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). Ec YqgF). Here, we reveal that Ec YqgF binds preferentially to branched DNA structures compared to single-stranded (ssDNA) and double-stranded DNA (dsDNA) , that the Ec YqgF: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 Ec YqgF 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 Ec YqgF has a DNA-independent, Mg2+-dependent 2+-dependent ATPase activity that is tightly coupled to DNA cleav- age. We have determined the crystal structure of Ec YqgF, 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 Ec YqgF variants, proficient in ATP-binding but not hydrol- ysis, which display a modest increase in the DNA-binding affinity, yet are devoid of endonucleolytic activ- ity, 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, Ec YqgF is func- tionally distinct. Importantly, the Ec YqgF dual activity that couples ATP hydrolysis to endonuclease activity is absent in RuvC. (c) 2025 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.