Chimeras of the homing endonuclease PI-SceI and the homologous Candida tropicalis intein: a study to explore the possibility of exchanging DNA-binding modules to obtain highly specific endonucleases with altered specificity.
Homing endonucleases are extremely specific endodeoxyribonucleases. In vivo, these enzymes confer mobility on their genes by inducing a very specific double-strand cut in cognate alleles that lack the cooling sequence for the homing endonuclease; the cellular repair of the double-strand break with the endonuclease-containing allele as a template leads to integration of the endonuclease gene, completing the homing process. As a result of their extreme sequence specificity, homing endonucleases are promising tools for genome engineering. For this purpose, it is desirable to design enzymes with defined new specificities. To analyse which DNA-binding elements are potential candidates for use in the design of enzymes with modified or even new specificity, we produced several chimeric proteins derived from the Saccharomyces cerevisiae VMA1 intein (PI-SceI) and the related Candida tropicalis VMA1 intein. Although the mature Candida intein is devoid of endonucleolytic activity, the exchange of two DNA-binding modules of PI-SceI with the homologous elements from the Candida intein results in an active endonuclease. The low sequence homology in these modules indicates that different protein-DNA contacts are responsible for the recognition of related DNA sequences. This flexibility in DNA recognition should, in principle, allow endonucleases to be produced with new specificities useful for genome engineering.
Steuer
Shawn
S
Justus-Liebig-Universität, Institut für Biochemie FB08, Heinrich-Buff-Ring 58, 35392 Giessen, Germany.
Pingoud
Vera
V
Pingoud
Alfred
A
Wende
Wolfgang
W
D000595:Amino Acid Sequence; D041022:Candida tropicalis / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific