Tyrosine 27 of the specificity polypeptide of EcoKI can be UV crosslinked to a bromodeoxyuridine-substituted DNA target sequence.
The specificity (S) subunit of the restriction enzyme EcoKI imparts specificity for the sequence AAC(N6)GTGC. Substitution of thymine with bromodeoxyuridine in a 25 bp DNA duplex containing this sequence stimulated UV light-induced covalent crosslinking to the S subunit. Crosslinking 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 subunit. Peptide sequencing of a proteolytically-digested, crosslinked complex identified tyrosine 27 in the S subunit as the site of crosslinking. This is consistent with the role of the N-terminal domain of the S subunit in recognizing the AAC sequence. 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 similar DNA contact in these other enzymes.
Chen
A
A
Institute of Cell and Molecular Biology, University of Edinburgh, UK.
Powell
L M
LM
Dryden
D T
DT
Murray
N E
NE
Brown
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001973:Bromodeoxyuridine; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D014443:Tyrosine; D004247:DNA; D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI; D014357:Trypsin; D001973:Bromodeoxyuridine