IDENTIFICATION OF CONTACTS BETWEEN TOPOISOMERASE-I AND ITS TARGET DNABY SITE-SPECIFIC PHOTO-CROSS-LINKING

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, binds and clea ves duplex DNA at sites containing the sequence 5'-(T/C)CCTT. We repor t the identification of Tyr70 as the site of contact between the enzym e and the +4C base of its target site. This was accomplished by UV-cro sslinking topoisomerase to bromocytosine-substituted DNA, followed by isolation and sequencing of peptide-DNA photoadducts. A model for the topoisomerase-DNA interface is proposed, based on the crystal structur e of a 9 kDa N-terminal tryptic fragment. The protein domain fits into the DNA major groove such that Tyr70 is positioned close to the +4C b ase and Tyr72 is situated near the +3C base. Mutational analysis indic ates that Tyr70 and Tyr72 contribute to site recognition during covale nt catalysis. We propose, based an this and other studies of the vacci nia protein, that DNA backbone recognition and reaction chemistry are performed by a relatively well-conserved 20 kDa C-terminal portion of the vaccinia enzyme, whereas discrimination of the DNA sequence at the cleavage site is accomplished by a separate N-terminal domain, which is less conserved between viral and cellular proteins. Division of fun ction among distinct structural modules may explain the different site specificities of the eukaryotic type I topoisomerases.