THE DOMAIN ORGANIZATION OF HUMAN TOPOISOMERASE-I

Using limited proteolysis, me show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence compar isons with other cellular Topo I enzymes. The enzyme is comprised of ( i) an NH2-terminal domain (similar to 24 kDa), which is known to be di spensable for activity, (ii) the core domain (similar to 54 kDa), (iii ) a linker region (similar to 3 kDa), and (iv) the COOH-terminal domai n (similar to 10 kDa), which contains the active site tyrosine. The hi ghly conserved core and COOH-terminal domains are resistant to proteol ysis, while the unconserved NH2-terminal and linker domains are sensit ive. Noncovalent binding of Topo I to plasmid DNA or to short duplex o ligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of t he NH2-terminal domain, When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sen sitive to proteolysis whether or not duplex DNA is present. The net po sitive charge of the linker domain suggests that at a certain point in catalysis the linker may bind directly to DNA. Further, we show that limited subtilisin cleavage can generate a mixture of 60-kDa core and similar to 10-kDa COOH-terminal fragments, which retain a level. of to poisomerase activity that is nearly equal to undigested control sample s, presumably because the two fragments remain associated after proteo lytic cleavage, Thus, despite its potential role in DNA binding, the l inker domain (in addition to the NH2-terminal domain) appears to be di spensable for topoisomerase activity, Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited pr oteolysis pattern of the vaccinia viral Topo I, indicating that the tw o enzymes belong to separate eukaryotic topoisomerase I subfamilies.