Abstract

This chapter describes a chromatographic method that allows the separation on a small scale of DNA polymerase from Saccharomyces cerevisiae, Pol α, Pol δ, and Pol ε at a crude stage of purification. With proper caution, these partially purified enzymes can then be used in replication and repair studies. To obtain any of the DNA polymerases in pure form, anion-exchange matrices are by far the most efficient in separating these three DNA polymerases. The order of elution from anion-exchange columns is reproducibly the same, that is, Pol δ elutes first, followed by Pol α, and Pol ε elutes last. In the absence of readily available specific antibodies to each of the DNA polymerases, a biochemical approach can be employed to determine their identities. For instance, both Pol β and Pol γ are resistant to aphidicolin and sensitive to the presence of dideoxynucleotides in the assay, but only Pol γ is fully active with 50 mM MgC12 in the assay. Pol α is the only enzyme that is inhibited by low levels of N2-(p-n-butylphenyl) dGTP (BuPhdGTP). DNA primase activity copurifies with Pol α, but the three nuclear RNA polymerases can also prime DNA synthesis. Only Polγ, Pol δ, and Pol ε, because of their associated 3' → 5'- exonuclease activity carry out efficient DNA synthesis on template primers with mismatched primer termini.

Original languageEnglish
Pages (from-to)49-62
Number of pages14
JournalMethods in enzymology
Volume262
Issue numberC
DOIs
StatePublished - 1995

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