DNA binding and recognition by the IIs restriction endonuclease MboII.
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 Ca(2+) is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca(2+), 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.
Soundararajan
Meera
M
Department of Biochemistry and Molecular Genetics, The University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom.
Chang
Zhiyuh
Z
Morgan
Richard D
RD
Heslop
Pauline
P
Connolly
Bernard A
BA
D002118:Calcium / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D008274:Magnesium / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism*; D010957:Plasmids
D009841:Oligonucleotides; D004247:DNA; C024413:endodeoxyribonuclease MboII; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D002118:Calcium