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Lookup NU author(s): Meera Soundararajan, Pauline Heslop, Professor Bernard Connolly
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The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca2+ is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca2+, possibly a (MboII)2-(DNA)2 complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)2-(DNA)2 complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.
Author(s): Heslop P; Connolly BA; Soundararajan M; Chang Z; Morgan RD
Publication type: Article
Publication status: Published
Journal: Journal of Biological Chemistry
Year: 2002
Volume: 277
Issue: 2
Pages: 887-895
ISSN (print): 0021-9258
ISSN (electronic): 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology, Inc.
URL: http://dx.doi.org/10.1074/jbc.M109100200
DOI: 10.1074/jbc.M109100200
PubMed id: 11606594
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