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DNA restriction dependent on two recognition sites: activities of the Sfi I restriction–modification system in Escherichia coli
Author(s) -
Bilcock Denzil T.,
Halford Stephen E.
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01266.x
Subject(s) - biology , restriction enzyme , plasmid , escherichia coli , dna , endonuclease , cleave , recognition sequence , restriction map , restriction fragment , microbiology and biotechnology , genetics , restriction site , gene
In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4–6 bp long, the Sfi I endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the Sfi I endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the Sfi I restriction–modification system were transformed with plasmids carrying Sfi I sites. The Sfi I system often failed to restrict the survival of a plasmid with one Sfi I site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated Sfi I sites were not restricted. No rearrangements of the plasmids carrying Sfi I sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, Sfi I displays all of the hallmarks of a restriction–modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro . For both restriction in vivo and DNA cleavage in vitro , Sfi I operates best with two recognition sites on the same DNA.