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.

Keywords: DNA digestion; MmeI; Restriction digestion; Restriction modification; Type IIS restriction enzymes.

Fig. 1

Comparison of cutting rates in the presence or absence of a DSB. (a) Gel showing a comparison of the amount of digestion among single DNA with one MmeI recognition site and similar sequences with a DSB between the recognition site and cut site. The DNA substrate with no DSB and an MmeI recognition site is 283 bps long. For the cutting across DSB experiment, the DNA molecule with no MmeI recognition site is 164 bps long, and the DNA molecule with an MmeI recognition site is 118 bps long. The percentage cut is shown below each lane. Percentages are based on the average of three runs, with a representative gel shown. (b) MmeI digestion of pairs of DNA substrates with 1-, 2-, or 3-base overhangs at 4 °C, 20 °C, and 37 °C. (c) Bar plot quantifying the percent digestion by MmeI shown in b. Error bars showing standard deviation calculated from three replicates (n = 3). (d) Visual representation of MmeI cutting with or without a DSB.