Purification of a DNA primase activity from the yeast Saccharomyces cerevisiae. Primase can be separated from DNA polymerase I.
A primase activity which permits DNA synthesis by yeast DNA polymerase I on a single-stranded circular phi X174 or M13 DNA or on poly(dT)n has been extensively purified by fractionation of a yeast enzyme extract which supports in vitro replication of the yeast 2-microns plasmid DNA (Kojo, H., Greenberg, B. D., and Sugino, A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7261-7265). Most of this DNA primase activity was separated from DNA polymerase activity, although a small amount remained associated with DNA polymerase I. The primase, active as a monomer, has a molecular weight of about 60,000. The primase synthesizes oligoribonucleotides of discrete size, mainly eight or nine nucleotides, in the presence of single-stranded template DNA and ribonucleoside 5'-triphosphates; it utilizes deoxyribonucleoside 5'-triphosphates as substrate with 10-fold lower efficiency. Product size, chromatographic properties, alpha-amanitin resistance, and molecular weight of the primase activity distinguish it from RNA polymerases I, II, and III. The DNA products synthesized by both primase and DNA polymerase I on a single-stranded DNA template were 200-500 nucleotides long and covalently linked to oligoribonucleotides at their 5'-ends. Addition of yeast single-stranded DNA-binding protein (Arendes, J., Kim, K. C., and Sugino, A. (1983) Proc. Natl. Acad. Sci. U.S. A. 80, 673-677) stimulated the DNA synthesis 2-3-fold.
Wilson
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Sugino
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D010584:Bacteriophage phi X 174 / Q000235:genetics; D002850:Chromatography, Gel; D004256:DNA Polymerase I / Q000302:isolation & purification*; D019915:DNA Primase; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004256:DNA Polymerase I