ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity.
Nuclear novobiocin binding proteins (NBPs) from a set of mouse L cells have been extensively purified by affinity chromatography on novobiocin-Sepharose columns. The NBPs, specifically eluted with 100 micrograms of novobiocin per ml, exhibited equivalent DNA topoisomerase activities (measured as ATP-dependent relaxation or catenation of phi X174 replicative-form I DNA substrate) when extracted from equal numbers of wild-type (WT-4) mouse L cells growing logarithmically at 34 degrees C or at 38.5 degrees C, from ts A1S9 cells similarly cultivated at the low, permissive temperature or from revertant ts+ AR cells in exponential growth at either temperature. The NBPs isolated from similar numbers of ts A1S9 cells grown to midlogarithmic phase and then incubated for 24 hr at 38.5 degrees C (the nonpermissive temperature) showed no topoisomerase II activity. Preliminary NaDodSO4/polyacrylamide gel electrophoretic analysis of enzymatically active material revealed that the NBPs of WT-4 and ts+ AR cells grown at 34 degrees C comprised three major polypeptides of 76,000, 74,000, and 30,000 daltons and a number of larger molecular mass components present in trace amounts. The NBP of ts A1S9 cells grown at the permissive temperature was similar, except that the 30,000-kilodalton polypeptide was not detected. Such enzymatically active NBPs from WT-4 and ts+ AR cells were unaffected by 100 micrograms of novobiocin per ml, whereas the analogous preparation from ts A1S9 cells was totally inhibited. On the basis of these and other considerations, it is postulated that the ts A1S9 locus of mouse L cells encodes a temperature-sensitive polypeptide that is required for normal DNA topoisomerase II activity.
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D007739:L Cells / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009675:Novobiocin / Q000378:metabolism*
D002352:Carrier Proteins; D009675:Novobiocin; D004250:DNA Topoisomerases, Type II