Homologous and heterologous protein-protein interactions of human DNA topoisomerase IIalpha.
DNA topoisomerase II (topo II; EC 5.99.1.3) is a nuclear enzyme whose DNA decatenating activity on newly replicated DNA is essential to successful cell division. Topo II catalytic activity proceeds by a concerted DNA breakage-reunion reaction coordinated between two interacting, homologous subunits. Human and yeast topo II have recently been shown to enter into heterologous protein-protein interactions and some of these interactions appear necessary for successful chromosomal segregation. In the present study, the sequences mediating homologous and heterologous protein-protein interactions have been investigated biochemically using various truncated peptides from the major alpha form of human topo II. From nonreducing gel electrophoresis and solid-phase protein-protein binding (Far Western) assays, topo II homodimerization appeared to be minimally governed by the region between amino acids 951 and 1042. However, maximal homodimerization and multimerization required sequences C-terminal to position 1042. Topo II peptides were also able to interact with 10-12 nuclear proteins from HeLa cells, termed topo II-interactive proteins or TIPs. Interestingly, small topo II peptides between residues 808 and 951 that did not homodimerize with topo II (857-1447) were nonetheless capable of binding to HeLa TIPs. These interactions were confirmed by use of topo II affinity chromatography for isolation of specific TIPs from HeLa nuclear extracts. Taken together, these data confirm that human topo II is also capable of heterologous interactions with nuclear proteins and that the region governing these interactions is distinct from, but has some overlap with, sequences directing topo II homodimerization.
Kroll
D J
DJ
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center and University of Colorado Cancer Center, Denver 80262, USA. david.kroll@uchsc.edu
D000951:Antigens, Neoplasm; D001665:Binding Sites; D002846:Chromatography, Affinity; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004250:DNA Topoisomerases, Type II