Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change

Abstract

Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4–8 bp long, with very few exceptions cleaving DNA more frequently. Tso I is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus -family exhibit ‘affinity star’ activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue—sinefungin (SIN). Here, we define a novel type of inherently built-in ‘star’ activity, exemplified by Tso I. The Tso I ‘star’ activity cannot be described under the definition of the classic ‘star’ activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The Tso I SCS comprises several degenerated variants of the cognate site. Although the efficiency of Tso I SCS cleavage is lower in comparison to the cognate Tso I recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The Tso I/SAC REase may serve as a novel tool for DNA manipulation. Electronic supplementary material The online version of this article (10.1007/s00253-019-09731-0) contains supplementary material, which is available to authorized users.

Noncanonical DNA Cleavage by BamHI Endonuclease in Laterally Confined DNA Monolayers Is a Step Function of DNA Density and Sequence

“…Cleavage at these sites can be promoted by varying parameters, including temperature, pH, or buffer composition, which results in an increase in the number of cuts in the DNA. This is usually an unwanted action of REases (termed "star activity" [3][4][5][6][7]) and has been shown to vary between isoschizomers (REases sharing the same cognate site) depending on structural differences affecting the DNA-binding mechanism [8].…”