Creation of a type IIS restriction endonuclease with a long recognition sequence.
Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5', four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.
Lippow
Shaun M
SM
Codon Devices, Inc, Cambridge, MA 02139, USA. slippow@gmail.com
Aha
Patti M
PM
Parker
Matthew H
MH
Blake
William J
WJ
Baynes
Brian M
BM
Lipovsek
Dasa
D
D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific