An auxiliary protein for DNA polymerase-delta from fetal calf thymus.
An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.
D000818:Animals; D002417:Cattle; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D011247:Pregnancy; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology*
D015636:Magnesium Chloride; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium