Background Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). Methods and results We used synthetic double-stranded oligos with MmeI recognition sites close to 5′ end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. Conclusion We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.
Background: Type IIS restriction enzymes cut outside of their recognition sites, allowing them to remove their own binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). Methods and Results: We used synthetic double-strand oligos with MmeI recognition sites close to 5′ end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the oligos are held together by transient base-pairing interactions between compatible overhangs. In this paper, we propose a model to explain this observation and characterize the nature of the brief interaction needed for digestion. Conclusion: We found that MmeI can cut across a DSB, and factors that increase the duration of the basepair interactions across the DSB (such as longer overhangs) or increase enzymatic activity (higher temperatures) tend to increase the amount of digestion. The ability of MmeI, and potentially other Type IIS enzymes, to cut across a DSB can result in DNA digestion without the recognition and target sites being ligated together.
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