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

Abstract

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.

ATP-dependent restriction enzyme SauUSI fromStaphylococcus aureusis also abona fidesingle strand DNA endonuclease

“…The copyright holder for this preprint this version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.11.519956 doi: bioRxiv preprint transient dsDNA formed by the ssDNA (8,(10)(11)(12)16). Here, we demonstrate that the restriction endonuclease SauUSI from Staphylococcus aureus, which is a sequence-specific dsDNA shredder (17) , is also a bona fide non-sequence-specific ssDNA endonuclease.…”

“…Depletion of NTPs and dNTPs during phage DNA replication and transcription will activate cleavage of ssDNA, including the replicating phage genome and that formed transiently in the host genome, causing abortive infection. If the foreign DNA entering the host bacteria is double-stranded and has the SauUSI target sites, shredding of the foreign dsDNA would be the prominent barrier to the entry (17).…”

“…Though, during the early days of research on restriction endonucleases, there were exciting reports of restriction endonucleases cleaving ssDNA (8–15), they could be explained as arising from cleavage of transient dsDNA formed by the ssDNA (8, 10–12, 16). Here, we demonstrate that the restriction endonuclease SauUSI from Staphylococcus aureus , which is a sequence-specific dsDNA shredder (17), is also a bona fide non-sequence-specific ssDNA endonuclease.…”

Characterization of BisI Homologs

“…EVE domain can be fused with PD-D/E xK nuclease and other nuclease domains such as Pin nuclease, PLD nuclease, and GIY-YIG family nucleases ( Bell et al, 2020 ). SauUSI is another example of Type IV REases with PLD nuclease-DNA helicase (ATPase)-Sra domain fusion that restricts hm5C/m5C modified DNA ( Xu et al, 2011 ; Tumuluri et al, 2021 ). It appeared that in nature, both MDREs with defined cleavage site (e.g., BisI and Dpn I) and those with undefined cleavage distance (McrBC, SauUSI, and GmrSD) have evolved in multiple occasions.…”

Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI

“…SauUSI is a Type IV restriction endonuclease present in Staphylococcus aureus that binds specifically to the methylated DNA target sequence 5'-S 5m CNGS-3' (S=C/G and N=A/T/G/C) (Xu et al, 2011). SauUSI has the ability to cleave DNA by two distinct mechanisms; they are: a) a highly efficient convergence-based cleavage mechanism when there are two or more target sequences, and b) a lesser efficient switchbased mechanism on DNA with only one target sequence (Tumuluri et al, 2021). Studies on the cleavage activity of SauUSI highlight its role in preventing the acquisition of exogenous genetic elements that have the ability to transform methicillin-resistant Staphylococcus aureus (MRSA) into vancomycinresistant Staphylococcus aureus (VRSA) (Corvaglia et al, 2010;Monk et al, 2012;Tumuluri et al, 2021).…”