A CATALYTIC DOMAIN OF EUKARYOTIC DNA TOPOISOMERASE-I

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'phosphotyrosyl)-enzyme intermediate, T he 314-amino acid vaccinia topoisomerase is the smallest member of thi s family and is distinguished from its cellular counterparts by its sp ecificity for cleavage at the target sequence 5'-CCCTT down arrow. Her e we show that Topo-(81-314), a truncated derivative that lacks the N- terminal domain, performs the same repertoire of reactions as the full -sized topoisomerase: relaxation of supercoiled DNA, site specific DNA transesterification, and DNA strand transfer. Elimination of the N-te rminal domain slows the rate of single-turnover DNA cleavage by 10(-3. 6), but has little effect on the rate of single-turnover DNA religatio n. DNA relaxation and strand cleavage by Topo(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are di splayed by a full-length mutant protein, Topo(Y70A/ Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact th e DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromi sed with respect to a rate limiting precleavage conformational step th at is contingent on DNA contacts made by Tyr-70 and Tyr-72.