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Reactions of type II restriction endonucleases with 8-base pair recognition sites
D T Bilcock1, L E Daniels, A J Bath
1Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
The Journal of Biological Chemistry
|December 14, 1999
Summary
Some type II restriction enzymes with 8-base pair recognition sites require two copies of the site for DNA cleavage, similar to SfiI. Others function independently at single or multiple sites.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Type II restriction endonucleases typically recognize 4-6 base pair (bp) DNA sequences.
- A few enzymes recognize 8-bp sites, which are rare and potentially less suitable for restriction.
- The enzyme SfiI, an 8-bp cutter, requires two recognition sites for DNA cleavage.
Purpose of the Study:
- To investigate whether other type II restriction endonucleases with 8-bp recognition sites also require two copies of their target sequence for cleavage.
- To characterize the cleavage mechanism of various 8-bp cutting endonucleases.
Main Methods:
- Tested several 8-bp recognition site endonucleases (SgfI, SrfI, FseI, PacI, PmeI, Sse8781I, SdaI, AscI, SgrAI) on plasmids containing either one or two copies of their respective recognition sequences.
- Compared cleavage rates and reaction mechanisms at single versus multiple sites.
Main Results:
- SgfI, SrfI, FseI, PacI, PmeI, Sse8781I, and SdaI cleaved DNA independently at each 8-bp site.
- AscI cleaved DNA at a single site as rapidly as at two sites but acted processively on the latter.
- SgrAI exhibited a preference for DNA with two sites, cleaving both in a concerted manner, similar to SfiI.
Conclusions:
- Restriction endonucleases requiring two 8-bp recognition sites for cleavage may be more common than previously thought.
- Despite the apparent inefficiency, these enzymes might play a significant role in DNA restriction in natural systems.