Mechanism of DNA cleavage by the endonuclease SauUSI: a major barrier to horizontal gene transfer and antibiotic resistance in Staphylococcus aureus

Acquisition of foreign DNA by Staphylococcus aureus, including vancomycin resistance genes, is thwarted by the ATP-dependent endonuclease SauUSI. Deciphering the mechanism of action of SauUSI could unravel the reason how it singularly plays a major role in preventing horizontal gene transfer (HGT) in S. aureus. Here, we report a detailed biochemical and structural characterization of SauUSI, which reveals that in the presence of ATP, the enzyme can cleave DNA having a single or multiple target site/s. Remarkably, in the case of multiple target sites, the entire region of DNA flanked by two target sites is shred into smaller fragments by SauUSI. Crystal structure of SauUSI reveals a stable dimer held together by the nuclease domains, which are spatially arranged to hydrolyze the phosphodiester bonds of both strands of the duplex. Thus, the architecture of the dimeric SauUSI facilitates cleavage of either single-site or multi-site DNA. The structure also provides insights into the molecular basis of target recognition by SauUSI. We show that target recognition activates ATP hydrolysis by the helicase-like ATPase domain, which powers active directional movement (translocation) of SauUSI along the DNA. We propose that a pile-up of multiple translocating SauUSI molecules against a stationary SauUSI bound to a target site catalyzes random double-stranded breaks causing shredding of the DNA between two target sites. The extensive and irreparable damage of the foreign DNA by shredding makes SauUSI a potent barrier against HGT.