Protein affinity chromatography with purified yeast DNA polymerase alpha detects proteins that bind to DNA polymerase.
We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase alpha polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase alpha matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase alpha accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast alpha or delta polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.
Miles
J
J
University of Utah School of Medicine, Department of Biochemistry, Salt Lake City 84132.
Formosa
T
T
D002846:Chromatography, Affinity / Q000379:methods; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D046911:Macromolecular Substances; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D005656:Fungal Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004257:DNA Polymerase II