PROTEOLYTIC FOOTPRINTING OF VACCINIA TOPOISOMERASE BOUND TO DNA

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, 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 by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease-resistant amino- and carboxyl-terminal structural domains flanking a protease sensitive ''hinge.'' The hinge region, 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 tripartite domain structure overall, A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state, The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex, Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively, Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation, The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase.