The type IIS restriction enzyme MmeI can cut across a double-strand break

Abstract

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.

Targeting the chromatin structural changes of antitumor immunity

“…Part of the experimental process is similar to the ChIP-loop experiment and the process can be summarized as follows: cells are immobilized with formalin; the genome is digested with restriction enzymes; the protein DNA complex is precipitated with antibodies specific to the target protein; a biotin labeled splice is added to the digested fragment (this splice has a specific cleavage site; such as MmeI); and a secondary ligation reaction is performed [ 224 , 236 ]. Then, an enzyme with a splice is used for digestion (such as MmeI) and the resulting product is coupled with a splice, before performing in-depth sequencing [ [237] , [238] , [239] ]. ChIA-PET can be used to identify the sites where the target protein interacts with DNA and further identify the genes that the target protein may regulate ( Fig.…”