PROTEOLYTIC FOOTPRINTING OF VACCINIA TOPOISOMERASE FOUND TO DNA

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme f amily, binds duplex DNA and forms a covalent protein . DNA complex at sites containing a conserved sequence element 5'-CCCTT down arrow. The structure of the enzyme in the free and DNA-bound states was probed b y limited proteolysis. The free topoisomerase (a 314-amino acid polype ptide) consists of protease-resistant amino- and carboxyl-terminal str uctural domains flanking a protease sensitive ''hinge.'' The hinge reg ion, located between residues 135 and 142, is defined by accessibility to three different proteases, The amino-terminal region is punctuated by a trypsin sensitive ''bridge'' at Arg-80, suggesting at least a tr ipartite domain structure overall, A specific subset of residues acces sible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state, The trypsin-sensitive site at Arg-80 is prote cted almost completely in the covalent complex, Within the hinge regio n, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotryp sin, and V8, respectively, Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation, The 20-kD a carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topo isomerase.