Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma.
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.
D000818:Animals; D004256:DNA Polymerase I / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D043211:Exodeoxyribonuclease V; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity
D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D003851:Deoxyribonucleases; D004720:Endonucleases; D005092:Exonucleases; D043211:Exodeoxyribonuclease V