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Activity and structure of EcoKMcrA
Honorata Czapinska1, Monika Kowalska1, Evelina Zagorskaite2
1International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Nucleic Acids Research
|August 15, 2018
Summary
Escherichia coli McrA (EcoKMcrA) is a restriction enzyme that cuts modified DNA. Researchers characterized its endonuclease activity, structure, and DNA binding, revealing how it recognizes specific DNA sequences.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Escherichia coli McrA (EcoKMcrA) is a restriction endonuclease.
- It recognizes and cleaves DNA modified with methylcytosine and hydroxymethylcytosine.
Purpose of the Study:
- To biochemically characterize EcoKMcrA.
- To determine its endonuclease activity, structure, and DNA binding mechanisms.
Main Methods:
- Biochemical assays
- Small-angle X-ray scattering (SAXS)
- X-ray crystallography
- Electrophoretic mobility shift assay (EMSA)
- Footprinting experiments
- Pyrrolocytosine fluorescence
Main Results:
- Demonstrated EcoKMcrA's endonuclease activity.
- Determined the crystal structure of EcoKMcrA, revealing dimer formation via C-terminal HNH domains and separate N-terminal DNA binding sites.
- N-terminal domains sense modified cytosines without base flipping.
- Enzyme activity requires Mn2+ and is sequence-context dependent.
- In vivo, EcoKMcrA restricts modified DNA, with activity dependent on the nuclease active site.
Conclusions:
- EcoKMcrA functions as a methylcytosine-dependent restriction endonuclease.
- Its structure and DNA binding properties enable sequence-specific recognition of modified DNA.
- The enzyme's activity is regulated by divalent cations and its active site.
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