Intrinsic Cleavage Inhibition by Thermophilic Type II-S Restriction Endonucleases

Restriction endonucleases (REs) are the cornerstone research tools used in molecular biology and biotechnology. These enzymes are essential to several ground-breaking achievements, including DNA cloning, gene editing, and genome mapping. A subset of these enzymes, Type II-S restriction endonucleases (IISREs), cleave double-stranded DNA (dsDNA) not governed by sequence specificity, but rather at a fixed distance from their recognition sequence. These enzymes maintain complete cleavage promiscuity, allowing utilization in techniques such as Golden Gate assembly, Serial Analysis of Gene Expression (SAGE), and Restriction Endonuclease Protection, Selection, and Amplification (REPSA). However, laboratories generated evidence that intrinsically cleavage refractory sequences exist. Understanding the pervasiveness of these sequences and their potential effects on the utilization of IISREs can be observed and analyzed through techniques such as REPSA presented for the first time in this study