I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.
Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.
Bell-Pedersen
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D
Molecular Genetics Program, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Quirk
S M
SM
Bryk
M
M
Belfort
M
M
D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease