Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta.
A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.
Shimizu
K
K
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Santocanale
C
C
Ropp
P A
PA
Longhese
M P
MP
Plevani
P
P
Lucchini
G
G
Sugino
A
A
D000818:Animals; D001483:Base Sequence; D004256:DNA Polymerase I / Q000235:genetics; D019951:DNA Polymerase beta; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008274:Magnesium; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D011189:Potassium Chloride; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D017931:DNA Primers; D019384:Nucleic Acid Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D011189:Potassium Chloride; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; C084892:POL4 protein, S cerevisiae; D008274:Magnesium