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Novikoff hepatoma stimulatory factor IV has been resolved from the DNA polymerase-beta on a single-stranded DNA-cellulose column and then purified to > 95% homogeneity on hydroxylapatite. A single band of Mr = 12,000 is found on sodium dodecyl sulfate-polyacrylamide gels. Addition of factor IV to a DNA synthesis reaction causes (i) an increase in initial velocity, (ii) a prolongation of linear synthesis, and (iii) an increase in extent of incorporation. In the absence of factor IV, the reaction reaches a plateau in approximately 1 h. Factor IV, added at this point, causes resumption of synthesis with kinetics similar to when factor IV was present from the start. When factor IV is present, synthesis is followed by DNA degradation, indicating nuclease activity. Factor IV is shown to be an exonuclease which hydrolyzes double-stranded substrates in both the 3' to 5' and 5' to 3' directions at similar rates. Factor IV interacts with the 3.3 S beta-polymerase forming an aggregate sedimenting at 4.1 S and containing both polymerase and exonuclease activities. Analysis of fractions containing a beta-polymerase . exonuclease complex on polyacrylamide gels suggests a stoichiometry of 1:1. The exonuclease shows a strong preference for double-stranded substrates and is most active on poly(dA-dT). It can hydrolyze chains containing either a 3'- or 5'-phosphoryl or a 5'- or 3'-hydroxyl terminus. The product of digestion is predominantly 5'-nucleoside monophosphates. The enzyme cannot hydrolyze di- or trinucleotides, lacks RNase-H activity, and will not liberate thymine dimers from UV-irradiated DNA. The exonuclease has an alkaline pH optimum and requires a divalent cation. Since the properties of this exonuclease are unlike those of previously described mammalian DNases, we have named this enzyme mammalian DNase V.

A temperature-sensitive single-stranded DNA-binding protein (SSB) has been purified from mutant Escherichia coli (ssb-1) cells by use of affinity chromatography on blue dextran-Sepharose. An altered amino acid sequence in the mutant protein is apparent in tryptic digests, confirming that the ssb mutation is in the structural gene. The mutant protein is less effective than the wild type in protecting single-stranded DNA from nuclease S1 digestion and in inhibiting DNA-dependent ATPases. The purified protein supports replication of phage G4 DNA in vitro at 30 degrees C, although higher levels of mutant protein, 4-fold higher than wild type, are needed to do so. The mutant protein becomes less active in supporting replication above 30 degrees C and becomes inactive at 42 degrees C within 1 min. Activity is restored upon return to 20 degrees C. Despite its temperature sensitivity in vivo and in vitro, the mutant binding protein can renature fully after exposure to 100 degrees C. Thus, the mutant protein is both heat-stable and functionally temperature-sensitive.

We have isolated and purified to homogeneity a novel single-stranded DNA-binding protein from the Novikoff hepatoma. This protein is distinguished from other eukaryotic DNA-binding proteins by binding weakly, but cooperatively, to single-stranded DNA, by its ability to partially destabilize a double helix at 37 degrees C, and by its ability to stimulate DNA polymerase beta. The protein exists as a globular monomer of Mr = 48,000 and is capable of binding 45-49 nucleotides. It does not form a complex with the polymerase, but binds the DNA template, allowing an increased rate and extent of DNA synthesis. The enhancement of synthesis is greatest with larger gap-sized templates and with low polymerase concentrations. The mechanism of stimulation is thought to be due largely to placing the template strand into a conformation that facilitates rapid polymerization rather than strand displacement in advance of the polymerase. This protein has been named SSB-48.

A single-stranded DNA-binding protein (SSB) affinity column was prepared by optimizing the coupling of Escherichia coli single-stranded DNA-binding protein to Affi-Gel 10. The bound SSB retained its ability to specifically bind single-stranded DNA. When nuclease-treated cell extracts were incubated with the SSB beads overnight at 4 degrees C, a major protein of Mr = 25,000 was bound. At shorter incubation times, two additional proteins of Mr = 32,000 and 36,000 were also detected. In the absence of nuclease treatment, eight additional proteins ranging from Mr = 14,000 to 160,000 also bound to the affinity column. The major Mr = 25,000 protein has been shown to be a folded chromosome-associated protein. Its binding to SSB is strongly enhanced by the addition of DNA polymerase III or DNA polymerase III holoenzyme.

A new DNA-dependent ATPase named ATPase IV has been purified to apparent homogeneity from Escherichia coli as a by-product of DNA polymerase III purification. The enzyme has a specific activity of 360 mumol of ATP hydrolyzed per min/mg of protein. The purified enzyme exists as monomer with a molecular weight of 81,000. It sediments in a glycerol gradient as a single species of 4.5 S. The enzyme has considerable activity at 0 degree C and has a Q10 of 3.8. In the presence of a DNA effector and magnesium ion, the enzyme will hydrolyze ATP, dATP, GTP, or dGTP to a nucleoside diphosphate plus orthophosphate with a Km of 0.20, 0.50, 0.60, and 1.30 mM, respectively. The guanine nucleotides, however, are only 25-35% as effective as substrates compared with the adenine nucleotides. ATPase IV shows strong substrate inhibition by ATP, but not dATP, above 0.2 mM. The polynucleotide effector requirement can be satisfied by either single-stranded or double-stranded DNA. The enzyme binds the effector very tightly with a Km of 3 X 10(-8) M (nucleotide) for G4 DNA. The enzyme is inhibited by E. coli single-stranded DNA-binding protein, a variety of ATP analogues and N-ethylmaleimide. The relationship of ATPase IV to DNA polymerase III holoenzyme is discussed.