Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme.
We have purified yeast DNA polymerase II to near homogeneity as a 145-kDa polypeptide. During the course of this purification we have detected and purified a novel form of DNA polymerase II that we designate as DNA polymerase II. The most highly purified preparations of DNA polymerase II are composed of polypeptides with molecular masses of 200, 80, 34, 30, and 29 kDa. Immunological analysis and peptide mapping of DNA polymerase II and the 200-kDa subunit of DNA polymerase II indicate that the 145-kDa DNA polymerase II polypeptide is derived from the 200-kDa polypeptide of DNA polymerase II. Activity gel analysis shows that the 145- and the 200-kDa polypeptides have catalytic function. The polypeptides present in the DNA polymerase II preparation copurify with the polymerase activity with a constant relative stoichiometry during chromatography over five columns and co-sediment with the activity during glycerol gradient centrifugation, suggesting that this complex may be a holoenzyme form of DNA polymerase II. Both forms of DNA polymerase II possess a 3'-5' exonuclease activity that remains tightly associated with the polymerase activity during purification. DNA polymerase II is similar to the proliferating cell nuclear antigen (PCNA)-independent form of mammalian DNA polymerase delta in its resistance to butylpheny-dGTP, template specificity, stimulation of polymerase and exonuclease activity by KCl, and high processivity. Although calf thymus PCNA does not stimulate the activity of DNA polymerase II on poly(dA):oligo(dT), possibly due to the limited length of the template, the high processivity of yeast DNA polymerase II on this template can be further increased by the addition of PCNA, suggesting that conditions may exist for interactions between PCNA and yeast DNA polymerase II.
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
R K
RK
Hamatake
H
H
Hasegawa
A B
AB
Clark
K
K
Bebenek
T A
TA
Kunkel
A
A
Sugino
D001482:Base Composition; D001483:Base Sequence; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011089:Polydeoxyribonucleotides; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013698:Templates, Genetic
D007527:Isoenzymes; D011089:Polydeoxyribonucleotides; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III