Nucleic Acids Res
- T. H. Koch
- 22,4947^*952. 43 Beaucage, L. and Caruthers, M.H…
The specificity (S) subuntt of the restriction enzyme EcoKl Imparts specificity for the sequence AAC(N6)GTGC. Substitution of thymine with bromodeoxyurldlne in a 25 bp DNA duplex containing this sequence stimulated UV light-induced covalent crosslinking to the S subunrt. Crosslinklng occurred only at the residue complementary to the first adenine in the AAC sequence, demonstrating a close contact between the major groove at this sequence and the S subunrt. Peptlde sequencing of a proteolyticallydlgested, crosslinked complex Identified tyrosine 27 in the S subunrt as the site of crosslinklng. This is consistent with the role of the N-terminal domain of the S subunrt In recognizing the AAC sequence. Tyrosine 27 is conserved in the S subuntts of the three type I enzymes that share the sequence AA in the trinuclectide component of their target sequence. This suggests that tyrosine 27 may make a similar DNA contact in these other enzymes.