Biochemical and structural characterization of a type iv restriction endonuclease sauusi

Abstract

Restriction modification enzymes are major barriers to horizontal gene transfer HGT in bacteria These enzymes are diverse in the nature of targets that they recognize their co factor requirements and mechanisms of action They are broadly classified as modification independent and modification dependent restriction enzymes SauUSI is a modification dependent restriction endonuclease and is known to thwart the acquisition of Vancomycin resistance genes in Staphylococcus aureus Here I report a detailed biochemical and structural characterization of SauUSI where in the presence of ATP the enzyme can cleave DNA having a single or multiple target site s Remarkably in substrates with two or more target sites the DNA region flanked by two target sites is shred into smaller fragments I also report the first crystal structure of SauUSI which reveals a stable dimer with the interface at the nuclease domains of the two protomers The architecture of SauUSI facilitates cleavage of both DNA with a single or multiple target site s and provides a molecular basis for target recognition ATP hydrolysis powers DNA translocation and we propose that in a DNA flanked by two target sites translocating SauUSI molecules pile up against a target site bound SauUSI molecule eventually leading to multiple double stranded DNA breaks causing irreparable DNA shredding The lesser efficient single site cleavage is attributed to a switch based mechanism I also report for the first time that an ATP dependent restriction endonuclease SauUSI functions as a bona fide single stranded DNA nuclease Interestingly this nucleolytic activity is target site and nucleotide independent Further SauUSI functions as an endonuclease on ssDNA because of its ability to cleave substrates with gaps and mismatch bubbles However this nuclease activity is restricted to ssDNA as ssRNA is refractory to cleavage by SauUSI This ssDNA endonucleolytic activity of SauUSI is downregulated by the presence of single stranded DNA binding protein and Adenine nucleotides such