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- Ingemarson, Rolf, 1954-
(author)
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Herpes simplex ribonucleotide reductase
- 1989
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Doctoral thesis (other academic/artistic)abstract
- In all bacterial, plant and animal cells, as well as in many viruses, genetic information resides in DNA (deoxyribonucleic acid). Replication of DNA is essential for proliferation, and DNA-containing viruses (such as herpesviruses) must carry out this process within the mammalian cells they infect. The enzyme ribonucleotide reductase catalyzes the first unique step leading to the production of the four deoxy-ribonucleotides used to make DNA. Each deoxyribonucleotide is produced by reduction of the corresponding ribonucleotide. After infection of a mammalian cell with herpes simplex virus (HSV) a new ribonucleotide reductase activity appears, which is distinct from the mammalian enzyme activity. This is due to induction of a separate, virally-encoded ribonucleotide reductase. Two monoclonal antibodies were raised against HSV (type 1) ribonucleotide reductase, and were found to bind but not neutralize its enzyme activity. One antibody recognized a larger (140 kD) protein and the other a smaller (40 kD) protein, suggesting the HSV 1 ribonucleotide reductase had a heterodimeric composition similar to that found in many other organisms. The 140 kD protein was sequentially degraded to 110 kD, 93 kD and 81 kD proteins by a host (Vero) cell-specific serine protease. Of these different proteolytic products, at least the 93 kD residue was enzymatically active, suggesting that part of the 140 kD protein may have functions unrelated to ribonucleotide reduction. The 140 and 40 kD proteins bound tightly to each other in a complex of the a2ß2 type, as shown by analytical glycerol gradient centrifugation. An assay system for functional small and large subunits of HSV 1 ribonucleotide reductase was developed, using two temperaturesensitive mutant viruses, defective in either the large (tsl207) or small (tsl222) subunits. Active holoenzyme was reconstituted both in vitro, by mixing extracts from cells infected with either mutant, and in vivo by coinfection of cells with both mutants. The gene encoding the small subunit of HSV 1 ribonucleotide reductase was cloned into an expression plasmid under control of a tac promoter. The recombinant protein was purified to homogeneity from extracts of transfected E. coli, and was active when combined with large subunit, as provided by extracts of tsl222- infected hamster (BHK) cells. The protein contained a novel tyrosyl free radical that spectroscopically resembled, but was distinguishable from, the active-site free radical found in either the E. coli or mammalian small subunits of ribonucleotide reductase. The gene encoding the large subunit of HSV 1 ribonucleotide reductase was also expressed in E. coli, using similar techniques. The recombinant large subunit was immunoprecipitated from extracts of transfected bacteria, and showed weak activity when combined with small subunit, provided by extracts of tsl20-infected hamster (BHK) cells.
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