PURIFICATION AND SUBSTRATE-SPECIFICITY OF A PHAGE-T4 INTRON-ENCODED ENDONUCLEASE

The T4 phage td intron-encoded endonuclease (I-Tev 1) cleaves the intron-deleted td gene (td-DELTA-I) 23 nucleotides upstream of the intron insertion site on the noncoding strand and 25 nucleotides upstream of this site on the coding strand, to generate a 2-base hydroxyl overhang in the 3' end of each DNA strand. I-Tev I-157, a truncated form in which slightly more than one third (88 residues) of the endonuclease is deleted, was purified to homogeneity and shown to possess endonuclease activity similar to that of I-Tev I, the full-length enzyme (245 residues). The minimal length of the td-DELTA-I gene that was cleaved by I-Tev I and I-Tev I-157 has been determined to be exactly 39 basepairs, from -27 (upstream in exon1) to +12 (downstream in exon2) relative to the intron insertion site. Similar to the full-length endonuclease, I-Tev I-157 cuts the intronless thymidylate synthase genes from such diverse organisms as Escherichia coli, Lactobacillus casei and the human. The position and nature of the in vitro endonucleolytic cut in these genes are homologous to those in td-DELTA-I. Point mutational analysis of the td-DELTA-I substrate based on the deduced consensus nucleotide sequence has revealed a very low degree of specificity on either side of the cleavage site, for both the full-length and truncated I-Tev I.