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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes
Mahesh K Chand1, Neha Nirwan1, Fiona M Diffin2
1Division of Biology, Indian Institute of Science Education and Research, Pune, India.
Nature Chemical Biology
|September 22, 2015
Summary
Complex DNA-cleaving enzymes called Type ISP restriction-modification (RM) enzymes use ATP to move along DNA. Their structure reveals a novel mechanism for DNA double-strand breaks, involving multiple nicks rather than simple dimerization.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Complex nuclease machines are poorly understood.
- Type ISP restriction-modification (RM) enzymes are single polypeptide enzymes that cleave DNA.
- Understanding their mechanism is crucial for molecular biology.
Purpose of the Study:
- To elucidate the mechanism of Type ISP restriction-modification (RM) enzymes.
- To determine the structure of a Type ISP enzyme-DNA complex.
- To investigate the DNA cleavage process by these enzymes.
Main Methods:
- X-ray crystallography (2.7-Å resolution) of a Type ISP enzyme-DNA complex.
- Single-molecule techniques.
- Biochemical assays.
- Product sequencing of single cleavage events.
Main Results:
- The crystal structure revealed ATPase and nuclease domains located upstream of translocation direction.
- Enzymes translocate without DNA looping, forming a collision complex with distal nuclease domains.
- A novel endonuclease model involving multiple stochastic strand-nicking events was proposed.
Conclusions:
- Type ISP RM enzymes employ a unique mechanism for DNA double-strand break production.
- The observed structure and translocation mechanism challenge simple dimerization models.
- A new model for DNA scission involving sequential nicking events is suggested.
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