Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization.
Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.
Sharf
R
R
Rappaport Institute for Research in the Medical Sciences, Haifa, Israel.
Weisman-Shomer
P
P
Fry
M
M
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; C056750:factor D (liver); D004259:DNA-Directed DNA Polymerase