The Domain Organization of Human Topoisomerase I Export

@article{citeulike:3740814, abstract = {Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH[(2)]-terminal domain ([IMG]\_7702\_tex2html\_wrap40.xbm">24 kDa), which is known to be dispensable for activity, (ii) the core domain ([IMG]\_7702\_tex2html\_wrap40.xbm">54 kDa), (iii) a linker region ([IMG]\_7702\_tex2html\_wrap40.xbm">3 kDa), and (iv) the COOH-terminal domain ([IMG]\_7702\_tex2html\_wrap40.xbm">10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH[(2)]-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH[(2)]-terminal domain. When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive 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 [IMG]\_7702\_tex2html\_wrap40.xbm">10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH[(2)]-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies. 10.1074/jbc.271.13.7602}, author = {Stewart, Lance and Ireton, Gregory C. and Champoux, James J.}, citeulike-article-id = {3740814}, citeulike-linkout-0 = {http://dx.doi.org/http://dx.doi.org/10.1074/jbc.271.13.7602}, citeulike-linkout-1 = {http://dx.doi.org/10.1074/jbc.271.13.7602}, doi = {http://dx.doi.org/10.1074/jbc.271.13.7602}, journal = {J. Biol. Chem.}, keywords = {coletta, domain, topoisomerase}, month = {March}, number = {13}, pages = {7602--7608}, posted-at = {2008-12-03 15:12:12}, priority = {2}, title = {The Domain Organization of Human Topoisomerase I}, url = {http://dx.doi.org/http://dx.doi.org/10.1074/jbc.271.13.7602}, volume = {271}, year = {1996} }

TY - JOUR ID - citeulike:3740814 L3 - citeulike-article-id:3740814 N2 - Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH[(2)]-terminal domain ([IMG]_7702_tex2html_wrap40.xbm">24 kDa), which is known to be dispensable for activity, (ii) the core domain ([IMG]_7702_tex2html_wrap40.xbm">54 kDa), (iii) a linker region ([IMG]_7702_tex2html_wrap40.xbm">3 kDa), and (iv) the COOH-terminal domain ([IMG]_7702_tex2html_wrap40.xbm">10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH[(2)]-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH[(2)]-terminal domain. When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive 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 [IMG]_7702_tex2html_wrap40.xbm">10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH[(2)]-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies. 10.1074/jbc.271.13.7602 IS - 13 JF - J. Biol. Chem. EP - 7608 TI - The Domain Organization of Human Topoisomerase I VL - 271 SP - 7602 KW - coletta KW - domain KW - topoisomerase AU - Stewart, Lance AU - Ireton, Gregory AU - Champoux, James PY - 1996/March// UR - http://dx.doi.org/http://dx.doi.org/10.1074/jbc.271.13.7602 ER -