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.