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- W2413335087 abstract "This chapter discusses the purification of mammalian polymerases, DNA polymerase δ. DNA polymerase δ is one of three DNA polymerases required for replication of genomic DNA in eukaryotic cells. The enzyme has been purified from a number of mammalian tissues and cells including calf thymus, rabbit bone marrow, human placenta, human HeLa cells, human 293 cells, and mouse lymphocytes. DNA polymerase δ is a heterodimer with subunits of approximately 125 and 48 kDa. The enzyme is essentially nonprocessive in the absence of the accessory proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) but highly processive in their presence. PCNA stimulation of the activity and processivity of DNA polymerase δ on a poly(dA) oligo(dT) template is a hallmark of this DNA polymerase and can be used to distinguish Pol δ from the other mammalian DNA polymerases, α, β, γ, and ɛ. In addition to DNA polymerase activity, DNA polymerase 6 has a 3' →5'-exonuclease activity, which serves a proofreading function. The protocol is designed to produce nearly homogeneous enzyme. The tissue is disrupted in a buffer containing a cocktail of protease inhibitors and the first two steps are carried out as quickly as possible to minimize proteolysis. DEAE-cellulose chromatography removes nucleic acids and separates DNA polymerase δ from PCNA, whereas phenyl-agarose chromatography separates the enzyme from most of the contaminating proteases. Phosphocellulose chromatography partially separates DNA polymerase δ from DNA polymerases α and ɛ and removes the remaining PCNA, which does not bind to phosphocellulose. The major separation of DNA polymerase from other mammalian DNA polymerases occurs on hydroxylapatite chromatography, as is evident by the large stimulation of activity on a poly (dA). oligo(dT) template by PCNA after this step. Highly purified DNA polymerase δ is stable for months or years when stored at -70° but rapidly loses activity on repeated freezing and thawing." @default.
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- W2413335087 date "1995-01-01" @default.
- W2413335087 modified "2023-09-23" @default.
- W2413335087 title "[9] Purification of mammalian polymerases: DNA polymerase δ" @default.
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- W2413335087 doi "https://doi.org/10.1016/0076-6879(95)62011-7" @default.
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