DnaX complex of Escherichia coli DNA polymerase III holoenzyme. Central role of tau in initiation complex assembly and in determining the functional asymmetry of holoenzyme.

The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, tau and gamma, were purified from extracts of strains carrying overexpressing plasmids mutated in their frameshifting sequences such that they produced only one subunit or the other. The purified subunits were used to reconstitute the tau and gamma complexes which were characterized by functional assays. The gamma complex-reconstituted holoenzyme required a stoichiometric excess of DNA polymerase III core, beyond physiological levels, for activity. The tau subunit stimulated the gamma complex 2-fold, but could not be used to reconstitute a holoenzyme with gamma complex and stoichiometric quantities of core. In the presence of adenosine 5'-O-(3'-thiotriphospate) (ATP gamma S), the DNA polymerase III holoenzyme behaves as an asymmetric dimer; it can form only initiation complexes with primed DNA in one-half of the enzyme (Johanson, K. O., and McHenry, C. S. (1984) J. Biol. Chem. 259, 4589-4595). An asymmetric distribution of two products of the dnaX gene, gamma and tau, has been postulated to underlie the asymmetry of holoenzyme. To provide a direct test for this hypothesis, we reconstituted holoenzyme containing only the gamma or tau DnaX proteins. We observed that, although gamma could function in the presence of ATP and high concentrations of DNA polymerase III core, it was nearly inert in the presence of ATP gamma S. In contrast, tau-containing holoenzyme behaved exactly like native holoenzyme in the presence of ATP gamma S. These results implicate tau as a key component required to reconstitute holoenzyme with native behavior and show that tau plays a key role in initiation complex formation. These results also show that gamma is not a necessary component, since all of the known properties of native holoenzyme can be reproduced with a 9-subunit tau-holoenzyme.

Strand displacement activity of the human immunodeficiency virus type 1 reverse transcriptase heterodimer and its individual subunits.

By using a DNA substrate with defined gap size, we found that human immunodeficiency virus type 1 reverse transcriptase (HIV-RT) was able to perform strand displacement DNA synthesis. This activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor C and second by Escherichia coli single-stranded DNA-binding protein, which together allow DNA polymerase delta to perform strand displacement DNA synthesis (Podust, V., and Hübscher, U. (1993) Nucleic Acids Res. 21, 841-846). 3'-Azido-2',3'-dideoxythymidine triphosphate inhibited displacement completely, indicating that DNA synthesis is required for this reaction. The HIV-RT p66 polypeptide alone could perform limited strand displacement DNA synthesis, whereas the HIV-RT p51 polypeptide was completely inactive, likely due to its inability to replicate extensively on a M13 DNA template. On the other hand the HIV-RT p51 polypeptide enhanced the strand displacement activity of the HIV-RT p66 subunit at a molar ratio of 4:1, mainly by chasing short products into longer ones. Furthermore, kinetic experiments after complementation of HIV-RT p66 with HIV-RT p51 indicated that HIV-RT p51 can restore rate and extent of strand displacement activity by HIV-RT p66 compared with the HIV-RT heterodimer p66/p51, suggesting a function of the 51-kDa polypeptide.

Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase.

Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66. We have produced both subunits in separate Escherichia coli strains using expression vectors. Stop codons were placed immediately after the codon for the carboxyl-terminal residue of the mature processed p51 and p66 subunits found in viral particles. Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E. coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage. That this occurred to an extent greater than 95% was confirmed by sequencing the purified subunits. Examination of the activities of the individual p51 and p66 subunits on a variety of templates and under solution conditions optimized for each subunit revealed a significant catalytic activity for the natural p51 subunit. This result contrasts to results reported earlier for many recombinant forms without the natural amino and/or carboxyl termini. As expected from earlier work, the optimal homopolymeric template for the p66 subunit was poly(rA). For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC). The p51 subunit is 13- to 50-fold less active on poly(rC). These findings are discussed in the context of our earlier hypothesis (McHenry, C. S. (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap. 5, Springer-Verlag, New York) that the HIV reverse transcriptase might be functionally asymmetric with distinct plus- and minus-strand polymerases.

Isolation of the glycoprotein of vesicular stomatitis virus and its binding to cell surfaces.

The glycoprotein (G) of vesicular stomatitis virus (VSV) was radiolabelled, extracted and purified so that its potential interaction with host cell surfaces could be studied. When BHK-21 cells were incubated with the radiolabelled virus glycoprotein, the virus component rapidly attached to the cell surface. The attachment was shown to be temperature-dependent adn saturated at approx. 3 X 10(5) molecules/cell. The omission of Mg2+ or Ca2+ from the incubation medium had little effect on the glycoprotein binding. Treating the isolated G protein and intact virions with neuraminidase did not significantly decrease their binding to BHK-21 cells. Pre-incubating cells with trypsin did not decrease the attachment of VSV virions nor the binding of purified G protein. Treating cells with phospholipase A or phospholipase C suggested that the binding of the glycoprotein and the intact virion might have been dissimilar. Unlabelled glycoprotein competitively inhibited binding of the labelled molecules although the presence of intact virions did not inhibit attachment of the G protein. Likewise, saturating amounts of the glycoprotein did not decrease binding of VSV to BHK-21 cells. These observations suggested that either the isolated glycoprotein bound to cell surface components that were distinct from the virion receptor or that the manner of the purified glycoprotein attachment differed from the G protein still associated with the intact virion. Chemical crosslinking and diagonal two-dimensional gel electrophoresis were used to identify and to compare the cell surface components responsible for glycoprotein and virion attachment.

Neuroblastoma cell fusion by a temperature-sensitive mutant of vesicular stomatitis virus.

A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 39 degrees C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39 degrees C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion.