Mouse ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. It has earlier been shown that the carboxyl-terminal part of the R2 protein is essential for subunit association to form the active enzyme complex. We now demonstrate that protein R2 gives rise to a number of sharp 1H NMR resonances, significantly narrower than the major part of the resonances. This line narrowing of certain resonances indicates segmental mobility in the molecule. In two-dimensional 1H TOCSY spectra of protein R2, cross-peak patterns from about 25 amino acid residues are visible. Most of these were assigned to the carboxyl-terminal part of the protein by comparisons with cross-peak patterns of oligopeptides corresponding to the carboxyl terminus of mouse R2 and to the patterns of a seven amino acid residue carboxyl-terminal truncated form of protein R2. These results and the magnitude of the chemical shifts of the assigned residues demonstrate that the carboxyl-terminal part of mouse R2 protein is highly mobile compared to the rest of the protein and essentially unstructured. When protein R1 is added to a solution of protein R2, the sharp resonances are broadened, suggesting that the mobility of the carboxyl-terminal tail of protein R2 is reduced. The possibility of making direct observations of subunit interaction in native and mutagenized R1/R2 proteins should allow discrimination between effects of amino acid replacements on the catalytic mechanism and effects on subunit interaction.

The vaccinia virus gene that encodes the small subunit of ribonucleotide reductase was localized to the Hindlll F fragment by using degenerate oligonucleotide probes. DNA sequencing revealed a leftward-reading open reading frame that predicted a protein of 37 kilodaltons whose amino acid sequence was much more homologous to the mouse and clam M2 sequences (-80%) than to the corresponding herpesvirus (-27%) or procaryotic (-19%) gene products. Vaccinia virus mutants selected for the ability to grow in high concentrations of a specific inhibitor of ribonucleotide reductase, hydroxyurea, amplified the M2 gene and harbored tandem arrays (2 to 15 copies) of the gene within the HindIII F region. RNA isolated at early times after infection with wild-type virus and probed with an internal fragment of the M2 gene indicated one major (1.2 kilobases) and two minor (4.0 and 2.1 kilobases) transcripts. Si nuclease analysis and primer extension experiments identified an RNA start site 12 nucleotides upstream of the putative initiation ATG codon.

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Evidence from prokaryotic systems suggests that enzymes of dNTP synthesis are organized near the DNA replication apparatus, allowing direct utilization of dNTPs at their sites of synthesis. To investigate whether similar interactions exist within a eukaryotic environment, we have prepared anti-idiotypic antibodies to the small subunit of vaccinia virus ribonucleotide reductase, and we used these antibodies to search for proteins that interact with this enzyme. This approach identified a 34-kDa viral phosphoprotein, which, like ribonucleotide reductase itself, is localized within infected cells at DNA replication sites. After expression of its structural gene in Escherichia cohi, the recombinant protein was purified and found (i) to bind tightly to single-stranded DNA and (ii) to stimulate enzymatic activity of vaccinia ribonucleotide reductase. These observations suggest a physical association between dNTP synthesis and DNA replication in this viral system.To what extent is the enzymatic machinery for DNA precursor biosynthesis linked to the DNA replication apparatus? In prokaryotic cells, high replicative chain growth rates and low affinities of replicative DNA polymerases for dNTPs suggest functional connections between dNTP biosynthesis and utilization, which could help maintain high local concentrations of dNTPs at replication sites (1, 2). Considerable evidence, mostly from T4 phage-infected Escherichia coli, supports the existence of enzyme complexes that maintain such linkages. Evidence has been presented for similar complexes in eukaryotic cells (ref. 3; reviewed in ref. 2). Multienzyme aggregates have been described, but direct linkage in vivo between dNTP synthesis and DNA replication has not been demonstrated (2). Any work involving isolated enzyme aggregates suffers from possible artifacts involving disaggregation of weakly or transiently associated complexes or involving nonspecific aggregation, which might occur after artificial rupture of cells. Accordingly, we are using different approaches to determine whether dNTP synthesis is linked to DNA replication in a eukaryotic environment.The system chosen is vaccinia virus-infected primate cells in culture. Vaccinia DNA replication occurs at cytoplasmic sites called virosomes. Therefore, viral DNA replication can be studied in some isolation from nuclear DNA metabolism of the host cell. Vaccinia virus encodes several enzymes of DNA metabolism, including ribonucleotide reductase, thymidine kinase, thymidylate kinase, dUTPase, DNA polymerase, and topoisomerase (4). rNDP reductase is of particular interest, because of its role in catalyzing the first reaction committed to DNA synthesis. Moreover, the structural genes for both large (R1) and small (R2) subunits of vaccinia rNDP reductase have been cloned and expressed in our laboratory, and both purified recombinant proteins are available in quantity (ref. 5; unpublished data). As in the mammalian or E. coli enzymes, both the R1 and R2 proteins are homodimers.The present approach is analysis of ant...

The nature of recessive mutations at the autosomal locus encoding the purine salvage enzyme adenine phosphoribosyl transferase (APRT) was analyzed in a highly malignant human tumor cell line (the colorectal carcinoma line SW620). Mutant strains resistant to the purine analog 8-azaadenine were obtained in two steps. The first step selection for partial drug resistance produced strains hemizygous for APRT as a result of high frequency loss of one allele. In the second step selection, low frequency base substitutions, small deletions, or insertions produced complete azaadenine resistance. Luria-Delbruck fluctuation analysis of each step of this process indicated that the rate of mutation resulting from allele loss was over 100-fold greater than the rate of mutation resulting from base substitution. There was no reproducible difference in the rate of loss of either of the two APRT alleles even though one maps to a rearranged chromosome. Similarly base substitution rates for the two alleles were not significantly different. Polymorphic loci surrounding APRT on chromosome 16 band q24 were lost together with the selected gene in all isolates while polymorphic loci on the short arm of the chromosome were retained. Thus the high frequency loss of APRT in SW620 appears to be the result of multilocus deletions. SW620 derivatives behaving as heterozygotes were also obtained in the first step selections, but these constituted only 5% of isolates.