Abstract
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.
Highlights
A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae
In the yeast Saccharomyces cereuisiue, several DNA polymerases have beenpurified and characterized biochemicallyand genetically.DNA polymerase I is normally purified as afour polypeptide complex consisting of the 180-kDa catalytic polypeptide, an 86-kDa polypeptide with unknown function, and the two DNA primase subunits
By cloning the genes encoding each of the polypeptides and subsequent genetical studies, it hasbeen established that DNA polymerase I is a DNA replicase in yeast [3,4,5]
Summary
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 shownto be associated with a 68kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCRl4C open-reading frame (identified on S. cereuisiae chromosome I11 and provisionally called P O L X ) , while no activity was detected in a strain where POLX was deleted. The former peak fractions were pooled (Fraction IV) and directly applied on a TonenHydroxylapatite (8 pm, Tonen Inc., Japan) FPLC column (HR 10/10) equilibrated with buffer P
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have