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.

Abstract:

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.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Exonuclease Activity, Kinetic Parameters, Nucleotide Incorporation, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Sce Pol alpha Hamatake RK1990 Molecular Weight 180kD
Sce Pol alpha Hamatake RK1990 3-5' Exonuclease (proofreading) No
Sce Pol alpha Hamatake RK1990 5-3' Exonuclease No
Sce Pol alpha Hamatake RK1990 Processivity 10bp
Sce Pol alpha Hamatake RK1990 Specific Activity 90.2 units/mg Technique: Polymerase assay (poly[d(AT)]
Sce Pol alpha Hamatake RK1990 Specific Activity 219 units/mg Technique: Polymerase Assay (calf thymus DNA)
Sce Pol alpha Hamatake RK1990 KM 63nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Sce Pol alpha Hamatake RK1990 KM 3.8uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Sce Pol II Hamatake RK1990 Molecular Weight 145kD
Sce Pol II Hamatake RK1990 3-5' Exonuclease (proofreading) Yes
Sce Pol II Hamatake RK1990 Full length or truncated Full length
Sce Pol II Hamatake RK1990 Processivity 100bp
Sce Pol II Hamatake RK1990 Specific Activity 87.4 units/mg Technique: Polymerase assay (poly[d(AT)]
Sce Pol II Hamatake RK1990 Specific Activity 176 units/mg Technique: Polymerase Assay (calf thymus DNA)
Sce Pol II Hamatake RK1990 KM 6.2uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Sce Pol II* Hamatake RK1990 Molecular Weight 200kD
Sce Pol II* Hamatake RK1990 3-5' Exonuclease (proofreading) Yes
Sce Pol II* Hamatake RK1990 5-3' Exonuclease No
Sce Pol II* Hamatake RK1990 Processivity 100bp
Sce Pol II* Hamatake RK1990 Specific Activity 243 units/mg Technique: Polymerase Assay (calf thymus DNA)
Sce Pol II* Hamatake RK1990 Specific Activity 333 units/mg Technique: Polymerase assay (poly[d(AT)]
Sce Pol II* Hamatake RK1990 KM 7.5uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Sce Pol II* Hamatake RK1990 KM 26nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Sce Pol III Hamatake RK1990 Molecular Weight 125kD
Sce Pol III Hamatake RK1990 3-5' Exonuclease (proofreading) Yes
Sce Pol III Hamatake RK1990 Specific Activity 45 units/mg Technique: Polymerase Assay (calf thymus DNA)
Sce Pol III Hamatake RK1990 Specific Activity 3.3 units/mg Technique: Polymerase assay (poly[d(AT)]

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