Acidic C terminus of vaccinia virus DNA-binding protein interacts with ribonucleotide reductase.

Abstract

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...

Molecular Genetic and Biochemical Characterization of the Vaccinia Virus I3 Protein, the Replicative Single-Stranded DNA Binding Protein

“…Interestingly, these were the only three mutants that were unable to support virus viability in vivo. Additionally, as stated above, it has been previously proposed that the acidic C-terminal 81 amino acids (aa) of I3 can mediate an interaction with the small subunit of the viral ribonucleotide reductase (RR) (6). The lesions within the I3-5 and -7 alleles fall within this domain, and the subtle changes introduced in these I3 proteins may be sufficient to disrupt the I3-RR interaction and may contribute to their inability to support viral replication in vivo.…”

“…Infected and mock-or siRNA-treated cells were harvested at the indicated time points (3,6,8,10,12, and 18 hpi) and subjected to Southern dot blot analysis as previously described (51). DNA was applied to Zeta Probe nitrocellulose (Bio-Rad) and detected using a radiolabeled probe representing the HindIII E and F fragments of the vaccinia virus genome; the bound probe was visualized by autoradiography and quantified on a PhosphorImager Storm Scanner using ImageQuant software.…”

Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the B1R protein kinase and phosphorylates in vitro the viral ssDNA-binding protein

“…It is known that the B1R protein is localized in cytoplasmic virus factories where viral replication occurs (Banham & Smith, 1992), and it is tempting to speculate that the binding of the ribosomal protein $2 kinase to the 36K protein may have the physiological function of directing the enzyme to this location. The viral ssDNA-binding phosphoprotein does appear to have the ability to associate with different proteins, as it was recently shown that it interacts with the vaccinia virus ribonucleotide reductase (Davis & Mathews, 1993). The finding that the ssDNA-binding phosphoprotein is most probably a substrate for the ribosomal protein S2/Sa kinase is interesting for another reason.…”

“…This phosphoprotein binds ssDNA cellulose and is eluted with 0.25 M-NaC1. Recently, it has been reported that it is encoded by the gene I3 of vaccinia virus (Davis & Mathews, 1993; S.C. Rochester & P. Traktman, personal communication).…”

Protein–DNA interactions in the T4 dNTP synthetase complex dependent on gene 32 single‐stranded DNA‐binding protein

“…At about the same time as we were making these observations, our laboratory discovered an SSB protein encoded by vaccinia virus, which interacts specifically with the virus‐coded ribonucleotide reductase (RNR) through its acidic C‐terminus (Davis and Mathews, 1993; I. Rajagopal, unpubl. data).…”