A. Chen et al., TYROSINE-27 OF THE SPECIFICITY POLYPEPTIDE OF ECOKI CAN BE UV CROSS-LINKED TO A BROMODEOXYURIDINE-SUBSTITUTED DNA TARGET SEQUENCE, Nucleic acids research, 23(7), 1995, pp. 1177-1183
The specificity (S) subunit of the restriction enzyme EcoKI imparts sp
ecificity for the sequence AAC(N-6)GTGC. Substitution of thymine with
bromodeoxyuridine in a 25 bp DNA duplex containing this sequence stimu
lated UV light-induced covalent crosslinking to the S subunit, Crossli
nking occurred only at the residue complementary to the first adenine
in the AAC sequence, demonstrating a close contact between the major g
roove at this sequence and the S subunit. Peptide sequencing of a prot
eolytically-digested, crosslinked complex identified tyrosine 27 in th
e S subunit as the site of crosslinking. This is consistent with the r
ole of the N-terminal domain of the S subunit in recognizing the AAC s
equence, Tyrosine 27 is conserved in the S subunits of the three type
I enzymes that share the sequence AA in the trinucleotide component of
their target sequence, This suggests that tyrosine 27 may make a simi
lar DNA contact in these other enzymes.