Human immunodeficiency virus 1 reverse transcriptase (RT) purified from virions is composed of a approximately 51,000 Mr polypeptide and a approximately 66,000 Mr polypeptide that are thought to be in heterodimer structure (Chandra et al., 1986; Hansen et al., 1988; Starnes & Cheng, 1989) and are identical except for a 15,000 Mr C-terminal truncation in the smaller species (Di Marzo-Veronese et al., 1986). We prepared individual bacterial-recombinant RTs as the approximately 66,000 Mr polypeptide (p66) or as the approximately 51,000 Mr polypeptide (p51) and then conducted various in vitro protein-protein binding experiments. Analytical ultracentrifugation studies in 0.25 M NaCl at pH 6.5 revealed that p66 was in monomer-dimer equilibrium with KA of 5.1 x 10(4) M-1. p51 failed to dimerize and behaved as a monomer under these conditions. Mixing of the p66 and p51 polypeptides resulted in a 1:1 heterodimer with KA of 4.9 x 10(5) M-1. These results on formation of the p66/p66 homodimer and p66/p51 heterodimer were confirmed by gel filtration analysis using FPLC Superose-12 columns. Binding between p66 and individual p66 segment polypeptides also was observed using an immunoprecipitation assay. Binding between p51 and p66 in this assay was resistant to the presence of approximately 1 M NaCl, suggesting that the binding free energy has a large hydrophobic component. C-Terminal truncation of p66 to yield a 29-kDa polypeptide eliminated binding to p66, and N-terminal truncation of p66 to yield a 15-kDa peptide also eliminated binding to p66. The results indicate that purified individual RT peptides p51 and p66 are capable of binding to form a 1:1 heterodimer and suggest that the central region of p66 is required for this subunit binding; the C-terminal region (15,000 Mr) of p66 appears to be required also, as p51 alone did not dimerize.

The coding region of a human beta-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with beta-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as beta-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural beta-polymerases. The purified enzyme was free of nuclease activity. We studied detailed catalytic properties of the recombinant beta-polymerase using defined template-primer systems. The results indicate that this beta-polymerase is essentially identical with natural beta-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG.

Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.

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Primer and dNTP recognition by purified HIV reverse transcriptase have been investigated. Earlier kinetic studies suggested that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence [Majumdar et al. (1988) J. Biol. Chem. 263, 15657-15665], and through use of a particularly high affinity template-primer analogue [r(I)n.Sd(C)28], rate values for formation of the first complex were calculated [Majumdar et al. (1989) Biochemistry 28, 1340-1346]. We now report rate values for first complex formation in the usual model replication system with poly[r(A)].oligo [d(T)] as template-primer. We find that 3'-azido-dTTP (AZTTP) is a linear competitive inhibitor of DNA synthesis against the substrate dNTP (dTTP) in the poly[r(A)].oligo[d(T)] replication system. This suggests that 3'-azido-dTTP and dTTP combine with the same form of the enzyme in the reaction scheme, i.e., the enzyme-primer complex. This is not trivial, since a second analogue, 3'-amino-dTTP, also is an inhibitor against dTTP, but the mechanism in this case is linear noncompetitive. Because the inhibition by 3'-azido-dTTP is linear competitive, the KD for physical binding to the enzyme is assumed to be the same as the Ki for inhibition (20 nM). Substrate kinetic studies of DNA synthesis using 3'-azido-dTTP as substrate revealed that the Michaelis constant is 3 microM. Therefore, the Km for this substrate analogue is 100-fold higher than the KD for binding of the analogue to the enzyme-primer complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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