Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

Abstract:

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).

Polymerases:

Topics:

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

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol alpha Weiser T1991 3-5' Exonuclease (proofreading) No
Human Pol alpha Weiser T1991 5-3' Exonuclease No
Human Pol alpha Weiser T1991 RNase H No
Human Pol alpha Weiser T1991 Processivity 450bp
Human Pol alpha Weiser T1991 Specific Activity 490 units/mg Technique: Polymerase Assay (M13 DNA)
Human Pol delta Weiser T1991 Molecular Weight 173kD
Human Pol delta Weiser T1991 3-5' Exonuclease (proofreading) Yes
Human Pol delta Weiser T1991 5-3' Exonuclease No
Human Pol delta Weiser T1991 3-5' Exo Specific Activity 451 units/mg
Human Pol delta Weiser T1991 Full length or truncated Full length
Human Pol delta Weiser T1991 RNase H No
Human Pol delta Weiser T1991 Processivity 1kb
Human Pol delta Weiser T1991 Specific Activity 132 units/mg Technique: Polymerase Assay (M13 DNA)
Human Pol epsilon Weiser T1991 3-5' Exonuclease (proofreading) Yes
Human Pol epsilon Weiser T1991 5-3' Exonuclease No
Human Pol epsilon Weiser T1991 3-5' Exo Specific Activity 426 units/mg
Human Pol epsilon Weiser T1991 Full length or truncated Full length
Human Pol epsilon Weiser T1991 RNase H No
Human Pol epsilon Weiser T1991 Processivity 1kb
Human Pol epsilon Weiser T1991 Specific Activity 176 units/mg Technique: Polymerase Assay (M13 DNA)

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