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The type IIS restriction enzyme MmeI can cut across a double-strand break
Maliha Tasnim1, T Jacob Selph1, Jason Olcott1
1Department of Cell Biology and Physiology, Brigham Young University, Provo, UT, 4005, 84602, USA.
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.
Insights
Type-IIS restriction enzymes like MmeI can cut across DNA double-strand breaks (DSBs). Their cutting efficiency depends on overhang length and temperature, enabling new molecular biology applications.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Type-IIS restriction enzymes cleave DNA outside their recognition sequences.
- This unique property facilitates applications like seamless cloning and CRISPR library generation.
- The enzyme MmeI recognizes TCCRAC and cuts 20 bp downstream.
Purpose of the Study:
- To investigate the capability of the Type-IIS restriction enzyme MmeI to cleave across a double-strand break (DSB).
Main Methods:
- Utilized synthetic double-stranded oligonucleotides with MmeI recognition sites near the 5' end.
- Varied overhang lengths, incubation times, and temperatures to assess digestion.
- Analyzed MmeI activity in the presence of a DSB.
Main Results:
- MmeI can bind and cut DNA across a DSB.
- Successful cutting occurs when compatible overhangs facilitate transient base-pairing, holding DNA edges together.
- The enzyme's binding and cutting sites can be located on opposite sides of the DSB.
Conclusions:
- MmeI demonstrates the ability to cut across double-strand breaks.
- Cutting efficiency is influenced by DNA overhang length and reaction temperature.
- Findings suggest potential for novel enzymatic applications involving DSBs.
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