Tyrosine 27 of the specificity polypeptide of EcoK\ can be UV crosslinked to a bromodeoxyuridine-substituted DNA target sequence

Abstract

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.

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Cite this paper

@inproceedings{Chen2005Tyrosine2O, title={Tyrosine 27 of the specificity polypeptide of EcoK\ can be UV crosslinked to a bromodeoxyuridine-substituted DNA target sequence}, author={Anlan Chen and Lynn M Powell and David T. F. Dryden and Noreen E. Murray and Tracie Brown}, year={2005} }