| Summary: | This study was carried out to investigate the existence of a functioning multi-enzyme complex, providing DNA precursors, in a mammalian cell system. Both uninfected BHK-21/C13 cells and cells infected with HSV-1 were investigated. Sucrose gradient centrifugation of uninfected BHK cell lysates showed co-sedimentation of a number of DNA precursor pathway enzymes, indicative of a multi-enzyme complex, including DNA polymerase, thymidine kinase, NDP kinase, dihydrofolate reductase and ribonucleotide reductase. This complex association was seen to be cell-cycle dependent and sensitive to ionic conditions. The enzymes involved were not non-specifically bound to either RNA or DNA, but did have template DNA associated at times of DNA replication. Sedimentation analysis of virus-induced enzymes synthesised in HSV-1 infected cells showed the enzymes not to form such a complete complex, although thymidine kinase and ribonucleotide reductase did sediment together. Again, this association was sensitive to ionic conditions. Optimally permeabilized cell systems were characterised and implemented to carry out kinetic analysis. No evidence for substrate channeling, in either uninfected or HSV-1 infected BHK-21/C13 cells, was obtained using such permeabilized cell systems.
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