TY - JOUR
T1 - DNA Polymerases from Saccharomyces Cerevisiae
AU - Burgers, Peter M.J.
N1 - Funding Information:
This work is dedicated to the memory of Severo Ochoa. This investigation has been aided by research grant 5R01 GM27242-14 from the National Institutes of Health, by grant PB90-0091 from Direcci6n General de lnvestigaci6n Cientifica y T6cnica, by grant BIOT CT 91-0268 from European Economic Community, and by an institutional grant from Fundaci6n Ram6n Areces.
PY - 1995
Y1 - 1995
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0028972928&partnerID=8YFLogxK
U2 - 10.1016/0076-6879(95)62008-7
DO - 10.1016/0076-6879(95)62008-7
M3 - Article
C2 - 8594373
AN - SCOPUS:0028972928
SN - 0076-6879
VL - 262
SP - 49
EP - 62
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -