Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities.
Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5' half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein-DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions.
Chan
Siu-hong
SH
New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
Bao
Yongming
Y
Ciszak
Ewa
E
Laget
Sophie
S
Xu
Shuang-yong
SY
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium